α-amylase mutants

ABSTRACT

The invention relates to a variant of a parent Termamyl-like a-amylase, which variant has a-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent a-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, Ca&lt;2+&gt; dependency and specific activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of 09/672,459, filed on Sep. 28, 2000 (now U.S. Pat. No. 6,436,888), which is a continuation of 09/182,859, filed on Oct. 29, 1998 (now U.S. Pat. No. 6,143,708), which is a continuation of PCT/DK97/00197 filed Apr. 30, 1997 which claims priority under 35 U.S.C. 119 of Danish applications 0515/96 filed Apr. 30, 1996, 0712/96 filed Jun. 28, 1996, 0775/96 filed Jul. 11, 1996, and 1263/96 filed Nov. 8, 1996, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, inter alia, to novel variants (mutants) of parent Termamyl-like a-amylases, notably variants exhibiting alterations in one or more properties (relative to the parent) which are advantageous with respect to applications of the variants in, in particular, industrial starch processing (e.g. starch liquefaction or saccharification).

BACKGROUND OF THE INVENTION

α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides, and there is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes.

Among more recent disclosures relating to α-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like α-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens α-amylase comprising the amino acid sequence shown in SEQ ID No. 4 herein and amino acids 301-483 of the C-terminal end of the B. licheniformis α-amylase comprising the amino acid sequence shown in SEQ ID No. 2 herein (the latter being available commercially under the tradename Termamyl™), and which is thus closely related to the industrially important Bacillus α-amylases (which in the present context are embraced within the meaning of the term “Termamyl-like α-amylases”, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens and B. stearothermophilus α-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like α-amylase, variants of the parent Termamyl-like α-amylase which exhibit altered properties relative to the parent.

BRIEF DISCLOSURE OF THE INVENTION

As indicated above, the present invention relates, inter alia, to novel α-amylolytic variants(mutants) of a Termamyl-like α-amylase, in particular variants exhibiting altered properties which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).

Alterations in properties which may be achieved in mutants of the invention are alterations in, e.g., substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile [such as increased stability at low (e.g. pH<6, in particular pH<5) or high (e.g. pH>9) pH values], stability towards oxidation, Ca²⁺ dependency, specific activity, and other properties of interest. For instance, the alteration may result in a variant which, as compared to the parent Termamyl-like α-amylase, has a reduced Ca²⁺ dependency and/or an altered pH/activity profile.

The invention further relates, inter alia, to DNA constructs encoding variants of the invention, to methods for preparing variants of the invention, and to the use of variants of the invention, alone or in combination with other α-amylolytic enzymes, in various industrial processes, e.g. starch liquefaction.

DETAILED DISCLOSURE OF THE INVENTION The Termamyl-like α-Amylase

It is well known that a number of α-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis α-amylase comprising the amino acid sequence shown in SEQ ID No. 2 (commercially available as Termamyl™) has been found to be about 89% homologous with the B. amyloliquefaciens α-amylase comprising the amino acid sequence shown in SEQ ID No. 4 and about 79% homologous with the B. stearothermophilus α-amylase comprising the amino acid sequence shown in SEQ ID No. 6. Further homologous α-amylases include an α-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the α-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31. Still further homologous α-amylases include the α-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the α-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis α-amylases are Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ (available from Genencor), and Keistase™ (available from Daiwa).

Because of the substantial homology found between these α-amylases, they are considered to belong to the same class of α-amylases, namely the class of “Termamyl-like α-amylases”.

Accordingly, in the present context, the term “Termamyl-like α-amylase” is intended to indicate an α-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl™, i.e. the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2 herein. In other words, a Termamyl-like α-amylase is an α-amylase which has the amino acid sequence shown in SEQ ID No. 2, No. 4 or No. 6 herein, or the amino acid sequence shown in SEQ ID No. 1 of WO 95/26397 (which amino acid sequence is shown in FIG. 1 and FIG. 2 herein) or in SEQ ID No. 2 of WO 95/26397 (which amino acid sequence is shown in FIG. 2 herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in FIG. 2 herein) or i) which displays at least 60%, such as at least 70%, e.g. at least 75%, or at least 80%, e.g. at least 85%, at least 90% or at least 95% homology with at least one of said amino acid sequences and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said α-amylases, and/or iii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified α-amylases which are apparent from SEQ ID Nos. 1, 3 and 5 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID Nos. 2, 4 and 6 herein, respectively), from SEQ ID No. 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in FIG. 1 herein and encodes the amino acid sequence shown in FIG. 1 herein) and from SEQ ID No. 5 of WO 95/26397, respectively.

In connection with property i), the “homology” may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG package version 7.3 (June 1993) using default values for GAP penalties [Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711].

Property ii) of the α-amylase, i.e. the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the relevant Termamyl-like α-amylase. The antibody, which may either be monoclonal or poly-clonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et. al., 1989. In this respect, immunological cross-reactivity between the α-amylases having the amino acid sequences SEQ ID Nos. 2, 4 and 6, respectively, has been found.

The oligonucleotide probe used in the characterization of the Termamyl-like α-amylase in accordance with property iii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the α-amylase in question. Suitable conditions for testing hybridization involve presoaking in 5×SSC and prehybridizing for 1 h at ˜40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 1000 μM ATP for 18h at ˜40° C., or other methods described by, e.g., Sambrook et al., 1989.

In the present context, “derived from” is intended not only to indicate an α-amylase produced or producible by a strain of the organism in question, but also an α-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an α-amylase which is encoded by a DNA sequence of synthetic and/or CDNA origin and which has the identifying characteristics of the α-amylase in question. The term is also intended to indicate that the parent α-amylase may be a variant of a naturally occurring α-amylase, i.e. a variant which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring α-amylase.

Parent Hybrid α-Amylases

The parent α-amylase may be a hybrid α-amylase, i.e. an α-amylase which comprises a combination of partial amino acid sequences derived from at least two α-amylases.

The parent hybrid α-amylase may be one which on the basis of amino acid homology and/or immunological cross-reactivity and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like α-amylase family. In this case, the hybrid α-amylase is typically composed of at least one part of a Termamyl-like α-amylase and part(s) of one or more other α-amylases selected from Termamyl-like α-amylases or non-Termamyl-like α-amylases of microbial (bacterial or fungal) and/or mammalian origin.

Thus, the parent hybrid α-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like α-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial α-amylase, or from at least one Termamyl-like and at least one fungal α-amylase. The Termamyl-like α-amylase from which a partial amino acid sequence derives may, e.g., be any of those specific Termamyl-like αa mylase referred to herein.

For instance, the parent α-amylase may comprise a C-terminal part of an α-amylase derived from a strain of B. licheniformis, and a N-terminal part of an α-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent α-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis α-amylase, and may, e.g. comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens α-amylase having the amino acid sequence shown in SEQ ID No. 4 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2, or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus α-amylase having the amino acid sequence shown in SEQ ID No. 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2.

The non-Termamyl-like α-amylase may, e.g., be a fungal α-amylase, a mammalian or a plant α-amylase or a bacterial α-amylase (different from a Termamyl-like α-amylase). Specific examples of such α-amylases include the Aspergillus oryzae TAKA α-amylase, the A. niger acid α-amylase, the Bacillus subtilis α-amylase, the porcine pancreatic α-amylase and a barley α-amylase. All of these α-amylases have elucidated structures which are markedly different from the structure of a typical Termamyl-like α-amylase as referred to herein.

The fungal α-amylases mentioned above, i.e. derived from A. niger and A. oryzae, are highly homologous on the amino acid level and generally considered to belong to the same family of α-amylases. The fungal α-amylase derived from Aspergillus oryzae is commercially available under the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like α-amylase (variant of the invention) is referred to—in a conventional manner—by reference to modification (e.g. deletion or substitution) of specific amino acid residues in the amino acid sequence of a specific Termamyl-like α-amylase, it is to be understood that variants of another Termamyl-like α-amylase modified in the equivalent position(s) (as determined from the best possible amino acid sequence alignment between the respective amino acid sequences) are encompassed thereby.

A preferred embodiment of a variant of the invention is one derived from a B. licheniformis α-amylase (as parent Termamyl-like α-amylase), e.g. one of those referred to above, such as the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2.

Construction of Variants of the Invention

The construction of the variant of interest may be accomplished by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant. The variant may then subsequently be recovered from the resulting culture broth. This is described in detail further below.

Altered Properties of Variants of the Invention

The following discusses the relationship between mutations which may be present in variants of the invention, and desirable alterations in properties (relative to those a parent, Termamyl-like α-amylase) which may result therefrom.

Decreased Ca²⁺ Dependency

It is highly desirable to be able to decrease the Ca²⁺ dependency of a Termamyl-like α-amylase. Accordingly, one aspect of the invention relates to a variant of a parent Termamyl-like α-amylase, which variant exhibits α-amylase activity and has a decreased Ca²⁺ dependency as compared to the parent α-amylase. Decreased Ca²⁺ dependency will in general have the functional consequence that the variant exhibits a satisfactory amylolytic activity in the presence of a lower concentration of calcium ion in the extraneous medium than is necessary for the parent enzyme. It will further often have the consequence that the variant is less sensitive than the parent to calcium ion-depleting conditions such as those obtained in media containing calcium-complexing agents (such as certain detergent builders).

Decreased Ca²⁺ dependency of a variant of the invention may advantageously be achieved, for example, by increasing the Ca²⁺ binding affinity relative to that of the parent Termamyl-like α-amylase; in other words the stronger the binding of Ca²⁺ in the enzyme, the lower the Ca²⁺ dependency.

It may be mentioned here that WO 96/23874 states that amino acid residues located within 10 Å from a sodium or calcium ion are believed to be involved in, or of importance for, the Ca²⁺ binding capability of the enzyme, and that in this connection the mutation N104D [of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2, or an equivalent (N to D) mutation of an equivalent position in another Termamyl-like α-amylase] is contemplated to be of particular interest with respect to decreasing the Ca²⁺ dependency of a Termamyl-like α-amylase.

Other mutations mentioned in WO 96/23874 as being of possible importance in connection with Ca²⁺ dependency include mutations which are contemplated therein to achieve increased calcium binding (and/or thermostability of the enzyme) via stabilization of the C-domain (as defined in WO 96/23874) of the three-dimensional structure of a Termamyl-like α-amylase via formation, for example, of cysteine bridges or salt bridges. Thus, WO 96/23874 discloses that the C-domain of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2 may be stabilized by introduction of a cysteine bridge between domain A and domain C (as defined in WO 96/23874) by introduction of the following mutations:

A349C+I479C and/or L346C+I430C.

WO 96/23874 likewise discloses that a salt bridge may be obtained by introduction of one or more of the following mutations in the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2:

N457D,E

N457D,E+K385R

F350D,E+I430R,K

F350D,E+I411R,K

and that the calcium site of Domain C may be stabilized by replacing the amino acid residues H408 and/or G303 with any other amino acid residue, in particular by introducing one of the substitutions:

H408Q,E,N,D and/or G303N,D,Q,E

which are contemplated to provide better calcium binding or protection from calcium depletion.

(similar mutations in equivalent positions of other Termamyl-like α-amylases being encompassed hereby).

Other substitution mutations (relative to B. licheniformis α-amylase, SEQ ID No. 2) which are disclosed in WO 96/23874 as being of apparent importance, inter alia, in the context of reducing calcium dependency include the following: R23K, H156Y, A181T, A209V, R214, G310D and P345 (or equivalent mutations in equivalent positions in another Termamyl-like α-amylase).

In the context of the present invention, further substitution mutations which appear to be of importance, inter alia, in relation to reduction of calcium dependency include the following mutations in Domain B (as defined in WO 96/23874):

A181E,D,Q,N,V (which appear to result in shielding of the outermost Ca²⁺ binding site in the junction region between Domain A and Domain B to some extent);

I201(bulkier amino acid), e.g. I201W,F,L (which appear to result in slight alterations in the geometry of the region in the immediate vicinity of the Ca²⁺—Na⁺—Ca²⁺ binding site(s) in the junction region between Domain A and Domain B, and in the geometry and/or size of a nearby hole/cavity); and

Y203E,Q (which are believed to result in stronger binding of the outermost Ca ²⁺ ion in its binding site in the junction region between Domain A and Domain B);

(or equivalent mutations in equivalent positions in another Termamyl-like α-amylase).

Altered pH Optimum (Altered pH/activity Profile)

WO 96/23874 discloses that it is contemplated to be possible to change the pH optimum of a Termamyl-like α-amylase, or the enzymatic activity thereof at a given pH, by changing the pKa of the active site residues, and that this may be achieved, e.g., by changing the electrostatic interaction or hydrophobic interaction between functional groups of amino acid side chains of the amino acid residue to be modified and of its close surroundings.

In the context of the present invention, it is believed on the basis of electrostatic considerations [see, e.g., M. K. Gilson, Current Opinion in Structural Biology 5 (1995) pp. 216-223; and B. Honig and A. Nicholls, Science 268 (1995) pp. 1144-1149; and references given therein] and hygroscopicity considerations in relation to the three-dimensional structure of the Termamyl-like α-amylase disclosed in WO 96/23874 that mutations of relevance, inter alia, for altering (increasing or decreasing) the pH optimum of a Termamyl-like α-amylase include the following mutations or equivalents thereof [referring here to the sequence of B. licheniformis α-amylase (SEQ ID NO 2)]:

Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T; I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E; V312R,K,D,E; F323H; D325N; N326K,H,D,L; H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,E,L; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D; Q360K,R,E; D365N; G371D,R;

Increased Stability at Low (Acidic) pH

In the context of the present invention, mutations (amino acid substitutions) of importance with respect to achieving increased stability at low pH appear to include mutations corresponding to the following mutations in the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2:

mutations at positions H68, H91, H247, R305, K306, H382, K389, H405, H406, H450 or R483;

the mutations:

H140Y;

H142Y;

H156Y;

H159Y;

H140D+H142R;

H140K+H142D; or

H142Y+H156Y

as well as combinations of any two or more of these mutations.

Increased Thermostability and/or Altered Temperature Optimum (Altered Temperature/activity Profile)

A further aspect of the invention relates to a variant of a parent Termamyl-like α-amylase, which variant is the result of one or more amino acid residues having been deleted from, substituted in or added to the parent α-amylase so as to achieve increased thermostability of the variant.

In may be mentioned that in relation to achieving increased thermostability, WO 96/23874 discloses that a particularly interesting variant of a Termamyl-like α-amylase comprises a mutation corresponding to one of the following mutations (using the numbering of the B. licheniformis α-amylase amino acid sequence shown in SEQ ID NO 2):

L61W,V,F;

Y62W;

F67W;

K106R,F,W;

G145F,W

I212F,L,W,Y,R,K;

S151 replaced with any other amino acid residue and in particular with F,W,I or L;

R214W;

Y150R,K;

F143W; and/or

R146W.

WO 96/23874 further discloses in this connection that mutations corresponding to one or more of the following mutations in the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2 are of interest in relation to achieving increased thermostability relative to that of the parent α-amylase:

L241I,F,Y,W; and/or

I236L, F,Y,W

L7F,I,W

V259F,I,L

F284W

F350W

F343W

L427F,L,W

V481,F,I,L,W

In the context of the present invention, it can be seen from an alignment of the amino acid sequences of α-amylases from various Bacillus species that B. licheniformis α-amylase and B. amyloliquefaciens α-amylase both contain an “insertion” of three amino acids relative to, e.g., B. stearothermophilus α-amylase.

From a model of the structure of B. licheniformis α-amylase built on the basis of the three-dimensional structure of the Termamyl-like α-amylase disclosed in WO 96/23784 (vide supra), taking into account the homology of B. licheniformis α-amylase to the Termamyl-like α-amylase in question, it can be seen that the above-mentioned “insertion” lies within a part of the structure denoted “loop 8” in WO 96/23784, making this loop bulkier in B. licheniformis α-amylase than in the Termamyl-like α-amylase and resulting in a loop that protrudes from the structure, thereby possibly destabilizing the structure. It is therefore contemplated that deletion of one or more amino acids in the region in question in B. licheniformis or B. amyloliquefaciens α-amylase will improve the thermostability of these α-amylases.

Especially interesting in this connection is deletion of three amino acids within the partial sequence from T369 to I377 (referring to the amino acid sequence of B. licheniformis α-amylase shown in SEQ ID No. 2), i.e. the partial sequence: T369-K370-G371-D372-S373-Q374-R375-E376-I377 (or the corresponding partial sequence in B. amyloliquefaciens α-amylase). In addition to such deletions, substitution of one or more of the undeleted amino acids within the latter partial sequence may also be advantageous.

Preferable deletions of three amino acids in the partial sequence from T369 to I377 (in the B. licheniformis α-amylase) are deletion of K370+G371+D372 (i.e. K370*+G371*+D372*) or deletion of D372+S373+Q374 (i.e. D372*+S373*+Q374*) (or equivalent deletions in the corresponding partial sequence in B. amyloliquefaciens α-amylase).

Another type of mutation which would appear to be of value in improving the thermostability of these α-amylases is substitution (replacement) of the entire partial amino acid sequence from T369 to I377 (referring to the sequence of the B. licheniformis α-amylase) with one of the following partial sequences of six amino acids (sequence numbering increasing from left to right): I-P-T-H-S-V; I-P-T-H-G-V; and I-P-Q-Y-N-I (or one of the same substitutions of the corresponding partial sequence in B. amyloliquefaciens α-amylase).

Other mutations which can apparently be of some importance in relation to achieving increased thermostability include amino acid substitutions at the following positions (referring to SEQ ID No. 2):

R169 (e.g. R169I,L,F,T);

R173 (especially R173I,L,F,T);

I201F;

I212F;

A209L,T; or

V208I

as well as combinations of any two or more of these mutations.

Increased Thermostability at Acidic pH and/or at Low Ca²⁺ Concentration

In the context of the invention, mutations which appear to be of particular relevance in relation to obtaining variants according to the invention having increased thermostability at acidic pH (pH<7) and/or at low Ca²⁺ concentration include mutations at the following positions (relative to B. licheniformis α-amylase, SEQ ID No. 2):

H156, N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264, N265

It may be mentioned here that N and E amino acid residues, respectively, at positions corresponding to N109 and E211, respectively, in SEQ ID No. 2 constitute amino acid residues which are conserved in numerous Termamyl-like α-amylases. Thus, for example, the corresponding positions of these residues in the amino acid sequences of a number of Termamyl-like α-amylases which have already been mentioned (vide supra) are as follows:

Termamyl-like α-amylase N position E position B. licheniformis (SEQ ID No. 2) N190 E211 B. amyloliquefaciens (SEQ ID No. 4) N190 E211 B. stearothermophilus (SEQ ID No. 6) N193 E210 Bacillus NCIB 12512 (WO 95/26397) N195 E212 Bacillus NCIB 12513 (WO 95/26397) N195 E212 “Bacillus sp. #707” (Tsukampto et al.) N195 E212

Mutations of these conserved amino acid residues appear to be very important in relation to improving thermostability at acidic pH and/or at low calcium concentration, and the following mutations are of particular interest in this connection (with reference to the numbering of the B. licheniformis amino acid sequence shown in SEQ ID No. 2):

H156Y,D

N172R,H,K

A181T

N188P

N190L,F

H205C

D207Y

A209L,T,V

A210S

E211Q

Q264A,E,L,K,S,T

N265A,S,T,Y

as well as any combination of two or more of these mutations.

An example of a particularly interesting double mutation in this connection is Q264S+N265Y.

Altered Cleavage Pattern

In the starch liquefaction process it is desirable to use an α-amylase which is capable of degrading the starch molecules into long, branched oligosaccharides, rather than an α-amylase which gives rise to formation of shorter, branched oligosaccharides (like conventional Termamyl-like α-amylases). Short, branched oligosaccharides (panose precursors) are not hydrolyzed satisfactorily by pullulanases, which are used after α-amylase treatment in the liquefaction process, but before addition of a saccharifying amyloglucosidase (glucoamylase). Thus, in the presence of panose precursors, the product mixture present after the glucoamylase treatment contains a significant proportion of short, branched, so-called limit-dextrin, viz. the trisaccharide panose. The presence of panose lowers the saccharification yield significantly and is thus undesirable.

Thus, one aim of the present invention is to arrive at a mutant α-amylase having appropriately modified starch-degradation characteristics but retaining the thermostability of the parent Termamyl-like α-amylase.

It may be mentioned here that according to WO 96/23874, variants comprising at least one of the following mutations are expected to prevent cleavage close to the branching point:

V54L,I,F,Y,W,R,K,H,E,Q

D53L,I,F,Y,W

Y56W

Q333W

G57 all possible amino acid residues

A52 amino acid residues larger than A, e.g. A52W,Y,L,F,I.

Increased Specific Activity

In a further aspect of the present invention, important mutations with respect to obtaining variants exhibiting increased specific activity appear to include mutations corresponding to the following mutations in the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID No. 2:

mutations (amino acid substitutions) at positions S187 (especially S187D) or Q264 (e.g. Q264R,K,S);

mutations (substitutions) at position Y290 (especially Y290E,K); the mutation V54I;

as well as combinations of any two or more of the latter mutations, or combinations of one, two or more of the latter mutations with the following multiple mutation:

A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I

General Mutations in Variants of the Invention

It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more proline residues present in the part of the α-amylase variant which is modified is/are replaced with a non-proline residue which may be any of the possible, naturally occurring non-proline residues, and which preferably is an alanine, glycine, serine, threonine, valine or leucine.

Analogously, it may be preferred that one or more cysteine residues present among the amino acid residues with which the parent α-amylase is modified is/are replaced with a non-cysteine residue such as serine, alanine, threonine, glycine, valine or leucine.

Furthermore, a variant of the invention may—either as the only modification or in combination with any of the above outlined modifications—be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID No. 2 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like α-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID No. 2 by an Arg.

It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in any of the variants described herein.

Methods for Preparing α-Amylase Variants

Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of α-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the α-amylase-encoding sequence will be discussed.

Cloning a DNA Sequence encoding an α-Amylase

The DNA sequence encoding a parent α-amylase may be isolated from any cell or microorganism producing the α-amylase in question, using various methods well known in the art. First, a genomic DNA and/or CDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the α-amylase to be studied. Then, if the amino acid sequence of the α-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify α-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known α-amylase gene could be used as a probe to identify α-amylase-encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying α-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming α-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for α-amylase, thereby allowing clones expressing the α-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by S. L. Beaucage and M. H. Caruthers (1981) or the method described by Matthes et al. (1984). In the phosphoroamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or CDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al. (1988).

Site-directed Mutagenesis

Once an α-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the α-amylase-encoding sequence, is created in a vector carrying the α-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into α-amylase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence in question, or within the whole gene.

WO 96/23874 discloses that in connection with achieving improved binding of a substrate (i.e. improved binding of a carbohydrate species, such as amylose or amylopectin) by a Termamyl-like α-amylase variant, modified (e.g. higher) substrate specificity and/or modified (e.g. higher) specificity with respect to cleavage (hydrolysis) of substrate, the following codon positions for the amino acid sequence shown in SEQ ID NO 2 (or equivalent codon positions for another parent Termamyl-like α-amylase in the context of the invention) appear to be particularly appropriate for targetting:

13-18

50-56

70-76

102-109

163-172

189-199

229-235

360-364

327-335

Improvement of Liquefaction Performance at Low pH and Low Calcium Ion Concentration

For an α-amylase to be used in a starch liquefaction process it is of particular interest that it be thermostable and able to function at low pH and low calcium concentrations. In order to improve these properties of a parent Termamyl-like α-amylase, in particular the B. licheniformis α-amylase or a variant or hybrid thereof, random mutagenesis (preferably by use of doped or spiked oligonucleotide primers) followed by appropriate selection of the resulting mutated enzymes may be performed. The direction of selection of regions to randomize and selection of doping are based primarily on stabilization of calcium ions already present, and on improvement in residue/residue or domain/domain electrostatic interactions at low pH. In addition, the regions which have been shown to include positions important for achieving good starch liquefaction performance may be selected.

In order to prepare a variant of a parent Termamyl-like α-amylase having the above properties, at least one of the following regions may advantageously be subjected to random mutagenesis (the numbering of the amino acid residues being as in SEQ ID No. 2):

Region Residue Description I: 153-163 Calcium region between domain A & B, also containing H156 II: 178-192 Calcium region between domain A & B III: 193-214 Calcium region between domain A & B, also also containing A209 IV: 232-237 Calcium region between domain A & B V: 297-308 Calcium region between domain A & C VI: 403-409 Calcium region between domain A & C VII: 428-435 Calcium region between domain A & C VIII: 131-136 Region containing H133 IX: 164-175 Region in contact with H133 region X: 262-278 Region containing Q264

Preferably, two, three or four of the above regions are subjected to random mutagenesis in the construction of a novel α-amylase variant of the invention. For instance, the following combinations of regions are suitably subjected to random mutagenesis:

VIII+IX

VIII+IX+II

II+III+IV

IV+I

Furthermore, it is preferred that the mutagenesis is carried out by use of doped or spiked oligonucleotides. The doping is preferably done so as to introduce amino acids contributing to improved stability at low pH and reduced calcium dependency at low pH of the resulting α-amylase variant. Furthermore, when selecting the doping scheme, the possibility of introducing Asn and Gln residues should generally be avoided, since Asn and Gln residues in general are associated with instability at low pH. Preferably, when a Pro residue can be inserted with potential benefits (e.g. as assessed from protein-structural considerations), the doping scheme is prepared to include a preference for introduction of a Pro residue.

The parent Termamyl-like α-amylase to be subjected to random mutagenesis according to the above principle may be any wild type α-amylase or a variant thereof containing one or more mutations. The parent may be a hybrid between at least two α-amylases as explained in further detail herein. Preferably, the parent α-amylase is a mutant of the B. licheniformis α-amylase having the sequence shown in SEQ ID No. 2 containing at least one mutation, and preferably multiple mutations. The parent α-amylase may alternatively be a hybrid α-amylase which contains at least a part of the B. licheniformis (SEQ ID No. 2) α-amylase. Specific examples of parent α-amylases suited to mutagenesis according to the above-described principles include: variants of the B. licheniformis (SEQ ID No. 2) α-amylase which contain at least one of, i.e. one, two, three, four or all five of, the mutations H156Y, A181T, N190F, A209V and Q264S; hybrid α-amylases which contain a part of the B. licheniformis (SEQ ID No. 2) α-amylase, preferably a C-terminal part thereof, such as amino acids 35-483 thereof, and a part of another Termamyl-like α-amylase such as B. amyloliquefaciens (SEQ ID No. 4) α-amylase, preferably an N-terminal part thereof such as the first 38 amino acid residues thereof.

In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased stability at low pH and at low calcium concentration relative to the parent, the method comprising:

(a) subjecting a DNA sequence encoding the parent Termamyl-like α-amylase to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and

(c) screening for host cells expressing a mutated α-amylase which has increased stability at low pH and low calcium concentration relative to the parent α-amylase.

Step (a) of the latter method of the invention is preferably performed using doped primers, as described in the working examples herein (vide infra).

Method of Performing Random Mutagenesis

The random mutagenesis of a DNA sequence encoding a parent α-amylase to be performed in accordance with step a) of the above-described method of the invention may conveniently be performed by use of any method known in the art.

For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents.

The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties.

When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the amylolytic enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase.

Preferably, the doping is carried out using “constant random doping”, in which the percentage of wild-type and mutation in each position is predefined. Furthermore, the doping may be directed to have a preference for the introduction of certain nucleotides, and thereby a preference for introduction of one or more specific amino acid residues. The doping may, e.g., be made so as to allow for the introduction of 90% wild type and 10% mutations in each position. An additional consideration in choice of doping scheme is genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program (see the working examples herein) which, inter alia, ensures that introduction of stop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent α-amylase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the amylolytic enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism.

The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent amylolytic enzyme. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.

In some cases it may be convenient to amplify the mutated DNA sequence prior to the expression step (b) or the screening step (c) being performed. Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme. Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gramnegative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.

Localized random mutagenesis: the random mutagenesis may advantageously be localized to a part of the parent α-amylase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.

The localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above, or any other suitable technique known in the art.

Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above.

With respect to the screening step in the above-mentioned method of the invention, this may conveniently performed by use of an assay as described in connection with Example 2 herein.

With regard to screening in general, a filter assay based on the following is generally applicable:

A microorganism capable of expressing the mutated amylolytic enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein-binding filter and on top of that a second filter exhibiting a low protein binding capability. The microorganism is located on the second filter. Subsequent to the incubation, the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms. The first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified.

The filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose. The topfilter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g. cellulose acetate or Durapore™. The filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity.

The enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity.

The detecting compound may be immobilized by any immobilizing agent e.g. agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.

α-Amylase activity is detected by Cibacron Red labelled amylopectin, which is immobilized on agarose. For screening for variants with increased thermal and high-pH stability, the filter with bound α-amylase variants is incubated in a buffer at pH 10.5 and 60° or 65° C. for a specified time, rinsed briefly in deionized water and placed on the amylopectin-agarose matrix for activity detection. Residual activity is seen as lysis of Cibacron Red by amylopectin degradation. The conditions are chosen to be such that activity due to the α-amylase having the amino acid sequence shown in SEQ ID No. 2 can barely be detected. Stabilized variants show, under the same conditions, increased colour intensity due to increased liberation of Cibacron Red.

For screening for variants with an activity optimum at a lower temperature and/or over a broader temperature range, the filter with bound variants is placed directly on the amylopectin-Cibacron Red substrate plate and incubated at the desired temperature (e.g. 4° C., 10° C. or 30° C.) for a specified time. After this time activity due to the α-amylase having the amino acid sequence shown in SEQ ID No. 2 can barely be detected, whereas variants with optimum activity at a lower temperature will show increase amylopectin lysis. Prior to incubation onto the amylopectin matrix, incubation in all kinds of desired media—e.g. solutions containing Ca²⁻, detergents, EDTA or other relevant additives—can be carried out in order to screen for changed dependency or for reaction of the variants in question with such additives.

Testing of Variants of the Invention

The testing of variants of the invention may suitably be performed by determining the starch-degrading activity of the variant, for instance by growing host cells transformed with a DNA sequence encoding a variant on a starch-containing agarose plate and identifying starch-degrading host cells. Further testing as to altered properties (including specific activity, substrate specificity, cleavage pattern, thermoactivation, pH optimum, pH dependency, temperature optimum, and any other parameter) may be performed in accordance with methods known in the art.

Expression of α-Amylase Variants

According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an α-amylase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an α-amylase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly-adenylation sequences operably connected to the DNA sequence encoding the α-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus α-amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the invention encoding an α-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989)).

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an α-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method of producing an α-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the α-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).

The α-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Industrial Applications

The α-amylase variants of this invention possesses valuable properties allowing for a variety of industrial applications. In particular, enzyme variants of the invention are applicable as a component in washing, dishwashing and hard-surface cleaning detergent compositions. Numerous variants are particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing. Conditions for conventional starch-conversion processes, including starch liquefaction and/or saccharification processes, are described in, e.g., U.S. Pat. No. 3,912,590 and in EP patent publications Nos. 252,730 and 63,909.

Production of sweeteners from starch: A “traditional” process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an α-amylase (e.g. Termamyl™) at pH values between 5.5 and 6.2 and at temperatures of 95-160° C. for a period of ap-prox. 2h. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase (e.g. AMGTM) and a debranching enzyme, such as an isoamylase or a pullulanase (e.g. Promozyme™). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95° C.), and the liquefying α-amylase activity is denatured. The temperature is lowered to 60° C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.

After the saccharification process the pH is increased to a value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucoseisomerase (such as Sweetzyme™).

At least 3 enzymatic improvements of this process could be envisaged. All three improvements could be seen as individual benefits, but any combination (e.g. 1+2, 1+3, 2+3 or 1+2+3) could be employed:

Improvement 1. Reduction of the Calcium Dependency of the Liquefying Alpha-amylase.

Addition of free calcium is required to ensure adequately high stability of the α-amylase, but free calcium strongly inhibits the activity of the glucoseisomerase and needs to be removed, by means of an expensive unit operation, to an extent which reduces the level of free calcium to below 3-5 ppm. Cost savings could be obtained if such an operation could be avoided and the liquefaction process could be performed without addition of free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like α-amylase which is stable and highly active at low concentrations of free calcium (<40 ppm) is required. Such a Termamyl-like α-amylase should have a pH optimum at a pH in the range of 4.5-6.5, preferably in the range of 4.5-5.5.

Improvement 2. Reduction of Formation of Unwanted Maillard Products

The extent of formation of unwanted Maillard products during the liquefaction process is dependent on the pH. Low pH favours reduced formation of Maillard products. It would thus be desirable to be able to lower the process pH from around pH 6.0 to a value around pH 4.5; unfortunately, all commonly known, thermostable Termamyl-like α-amylases are not very stable at low pH (i.e. pH<20 6.0) and their specific activity is generally low.

Achievement of the above-mentioned goal requires a Termamyl-like α-amylase which is stable at low pH in the range of 4.5-5.5 and at free calcium concentrations in the range of 0-40 ppm, and which maintains a high specific activity.

Improvement 3.

It has been reported previously (U.S. Pat. No. 5,234,823) that when saccharifying with A. niger glucoamylase and B. acidopullulyticus pullulanase, the presence of residual α-amylase activity from the liquefaction process can lead to lower yields of dextrose if the α-amylase is not inactivated before the saccharification stage. This inactivation can typically be carried out by adjusting the pH to below 4.3 at 95° C., before lowering the temperature to 60° C. for saccharification.

The reason for this negative effect on dextrose yield is not fully understood, but it is assumed that the liquefying α-amylase (for example Termamyl™ 120 L from B. licheniformis) generates “limit dextrins” (which are poor substrates for B. acidopullulyticus pullulanase) by hydrolysing 1,4-α-glucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build-up of the trisaccharide panose, which is only slowly hydrolysed by glucoamylase.

The development of a thermostable α-amylase which does not suffer from this disadvantage would be a significant process improvement, as no separate inactivation step would be required.

If a Termamyl-like, low-pH-stable α-amylase is developed, an alteration of the specificity could be an advantage needed in combination with increased stability at low pH.

The methodology and principles of the present invention make it possible to design and produce variants according to the invention having required properties as outlined above. In this connection, particularly interesting mutations are mutations in a Termamyl-like α-amylase [for example Termamyl™ itself (B. licheniformis α-amylase; SEQ ID No. 2); or a Termamyl-like α-amylase having an N-terminal amino acid sequence (i.e. the partial sequence up to the amino acid position corresponding to position 35 in Termamyl™) which is identical to that in B. amyloliquefaciens α-amylase (SEQ ID No. 4), i.e. a Termamyl-like α-amylase having the following N-terminal sequence relative to amino acid sequence of Termamyl™:

A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I, where an asterisk(*)indicates deletion of the amino acid residue in question] at positions corresponding to any of the following positions in Termamyl™:

H133

H156

A181

A209

G310

H450

V128

N104

V54

S187

H293

A294

(where each of the latter amino acid residues may be replaced by any other amino acid residue, i.e. any other residue chosen among A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V), as well as the following triple deletions:

K370*+G371*+D372*

D372*+S373*+Q374*

Particularly preferred substitutions at the above-indicated positions are the following:

H133I

H156Y

A181T

A209V

G310D

H450Y

V128E

N104D

V54W,Y,F,I,L

S187D

H293Y

A294V.

Any combination of one or more (i.e. one, two, three, four . . . etc.) of the above indicated mutations may appropriately be effected in a Termamyl-like α-amylase in the context in question, and particularly interesting variants of the invention in the context of achieving one or more of the above-mentioned improvements in relation to the starch liquefaction behaviour of α-amylases include variants comprising combinations of multiple mutations corresponding to the following combinations of mutations in Termamyl™ (SEQ ID No. 2) itself:

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A 294V+K370*+G371*+D372*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54W+S187D+H293Y+A 294V+D372*+S373*+Q374*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A 294V+K370*+G371*+D372*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54Y+S187D+H293Y+A 294V+D372*+S373*+Q374*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A 294V+K370*+G371*+D372*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54F+S187D+H293Y+A 294V+D372*+S373*+Q374*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A 294V+K370*+G371*+D372*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54I+S187D+H293Y+A 294V+D372*+S373*+Q374*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A 294V+K370*+G371*+D372*;

H133I+H156Y+A181T+A209V+G310D+H450Y+V128E+N104D+V54L+S187D+H293Y+A 294V+D372*+S373*+Q374*;

Further interesting variants of the invention in this context include variants comprising single or multiple mutations corresponding to the following single or multiple mutations in Termamyl™ itself:

mutations (amino acid substitutions) at positions N172 (e.g. N172R,K), S187 (e.g. S187D), N188 (e.g. N188P), N190 (e.g. N190L,F), H205 (e.g. H205C), D207 (e.g. D207Y), A210 (e.g. A210S), Q264 (e.g. Q264S) or N265 (e.g. N265Y);

the following multiple mutations;

H156Y+A181T+A209V;

H156Y+A181T+N190F+A209V+Q264S

A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+A2 09V;

A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V; or

A1*+N2*+L3V+M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35I+H156Y+A181T+N1 90F+A209V+Q264S

as well as combinations of any two or more of the latter single or multiple mutations.

As already indicated, numerous variants according to the invention are particularly well suited for use in starch conversion, e.g. in starch liquefaction. In this connection, a further aspect of the present invention relates to compositions comprising a mixture of:

(i) the α-amylase from B. licheniformis having the sequence shown in SEQ ID No. 2 with one or more variants (mutant α-amylases) according to the invention derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID No. 6; or

(ii) the α-amylase from B. stearothermophilus having the sequence shown in SEQ ID No. 6 with one or more variants (mutant α-amylases) according to the invention derived from one or more other parent Termamyl-like α-amylases (e.g. from the B. licheniformis α-amylase having the sequence shown in SEQ ID No. 2, or from one of the other parent Termamyl-like α-amylases specifically referred to herein); or

(iii) one or more variants (mutant α-amylases) according to the invention derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID No. 6 with one or more variants (mutant α-amylases) according to the invention derived from one or more other parent Termamyl-like α-amylases (e.g. from the B. licheniformis α-amylase having the sequence shown in SEQ ID No. 2, or from one of the other parent Termamyl-like α-amylases specifically referred to herein).

Preferred mutations in a variant of B. stearothermophilus α-amylase to be incorporated in such a mixture include substitutions at N193 and/or at E210, and/or the pairwise deletions R179*+G180* or I181*+G182* (using the numbering of the amino acid sequence for this particular α-amylase).

Compositions of one of the latter types, containing B. stearothermophilus α-amylase or a variant thereof according to the invention, appear to have great potential for use in starch liquefaction. The ratio (expressed, e.g., in terms of mg of active amylolytic protein per liter of liquid medium) between the individual α-amylolytic components of a given mixture will depend on the exact nature and properties of each component.

Detergent Compositions

As mentioned above, variants of the invention may suitably be incorporated in detergent compositions. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.

Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another α-amylase.

α-Amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of α-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.

The present invention is further described with reference to the appended drawing, in which:

FIG. 1 shows the DNA sequence, together with the stop codon TAA, encoding the Bacillus strain NCIB 12512 α-amylase described in WO 95/26397, together with the amino acid sequence of the encoded α-amylase (cf. FIG. 2).

FIG. 2 is an alignment of the amino acid sequences of four parent Termamyl-like α-amylases in the context of the invention. The numbers on the extreme left designate the respective amino acid sequences as follows:

1: the amino acid sequence of the Bacillus strain NCIB 12512 α-amylase described in WO 95/26397;

2: the amino acid sequence of the Bacillus strain NCIB 12513 α-amylase described in WO 95/26397;

3: the amino acid sequence of the B. stearothermophilus α-amylase as shown in SEQ ID No. 6 herein;

4: the amino acid sequence of the Bacillus sp. #707 α-amylase described by Tsukamoto et al. in Biochem. Biophys. Res. Commun. 151 (1988), pp.25-31.

The numbers on the extreme right of the figure give the running total number of amino acids for each of the sequences in question. Note that for the sequence numbered 3 (corresponding to the sequence in SEQ ID No. 6), the alignment results in “gaps” at the positions corresponding to amino acid No. 1 and No. 175, respectively, in the sequences numbered 1, 2 and 4.

FIG. 3 illustrates the PCR strategy employed in Example 2 (vide infra).

MATERIALS AND METHODS Construction of pSNK101

This E. coli/Bacillus shuttle vector can be used to introduce mutations without expression of α-amylase in E. coli and then be modified in such way that the α-amylase is active in Bacillus. The vector was constructed as follows: The α-amylase gene in the pX vector (pDN1528 with the following alterations within amyL: BAN(1-33), H156Y, A181T, N190F, A209V, Q264S, the plasmid pDN1528 is further described in Example 1) was inactivated by interruption in the PstI site in the 5′ coding region of the alpha-amylase gene by a 1.2 kb fragment containing an E. coli origin fragment. This fragment was amplified from the pUC19 (GenBank Accession #:XO2514) using the forward primer: 5′-gacctgcagtcaggcaacta-3′ and the reverse primer: 5′-tagagtcgacctgcaggcat-3′. The PCR amplicon and the pX plasmid containing the α-amylase gene were digested with PstI at 37° C. for 2 hrs. The pX vector fragment and the E. coli origin amplicon were ligated at room temperature for 1 h and transformed in E. coli by electrotransformation. The resulting vector is designated pSnK101.

Fermentation and Purification of α-Amylase Variants

A B. subtilis strain harbouring the relevant expression plasmid is streaked on a LB-agar plate with 15 μg/ml chloramphenicol from −80° C. stock, and grown overnight at 37° C. The colonies are transferred to 100 ml BPX media supplemented with 15 μg/ml chloramphenicol in a 500 ml shaking flask.

Composition of BPX medium: Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodium caseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂0 9 g/l Pluronic ™ 0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 min. Afterwards the supernatant is filtered to obtain a completely clear solution. The filtrate is concentrated and washed on a UF-filter (10000 cut off membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3M NaCl over 6 column volumes. The fractions which contain the activity (measured by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and remaining color was removed by a treatment with 0.5% W/vol. active coal in 5 min.

Assay for α-Amylase Activity

α-Amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloured starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The α-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolysed by the α-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the α-amylase activity.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given α-amylase will hydrolyse a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure α-amylase protein) of the α-amylase in question under the given set of conditions.

General Method for Random Mutagenesis by use of the DOPE Program

The random mutagenesis may be carried out by the following steps:

1. Select regions of interest for modification in the parent enzyme

2. Decide on mutation sites and nonmutated sites in the selected region

3. Decide on which kind of mutations should be carried out, e.g. with respect to the desired stability and/or performance of the variant to be constructed

4. Select structurally reasonable mutations.

5. Adjust the residues selected by step 3 with regard to step 4.

6. Analyze by use of a suitable dope algoritm the nucleotide distribution.

7. If necessary, adjust the wanted residues to genetic code realism (e.g. taking into account constraints resulting from the genetic code (e.g. in order to avoid introduction of stop codons))(the skilled person will be aware that some codon combinations cannot be used in practice and will need to be adapted)

8. Make primers

9. Perform random mutagenesis by use of the primers

10. Select resulting α-amylase variants by screening for the desired improved properties.

Suitable dope algorithms for use in step 6 are well known in the art. One algorithm is described by Tomandl, D. et al., Journal of Computer-Aided Molecular Design, 11 (1997), pp. 29-38). Another algorithm, DOPE, is described in the following:

The Dope Program

The “DOPE” program is a computer algorithm useful to optimize the nucleotide composition of a codon triplet in such a way that it encodes an amino acid distribution which resembles most the wanted amino acid distribution. In order to assess which of the possible distributions is the most similar to the wanted amino acid distribution, a scoring function is needed. In the “Dope” program the following function was found to be suited: ${s \equiv {\prod\limits_{i = 1}^{N}\quad \left( \frac{\left( {x_{i}^{y_{i\quad}}\left( {1 - x_{i}} \right)} \right)^{1 - y_{i}}}{\left( {y_{i}^{y_{i}}\left( {1 - y_{i}} \right)} \right)^{1 - y_{i}}} \right)^{w_{i}}}},$

where the x_(i)'s are the obtained amounts of amino acids and groups of amino acids as calculated by the program, y_(i)'s are the wanted amounts of amino acids and groups of amino acids as defined by the user of the program (e.g. specify which of the 20 amino acids or stop codons are wanted to be introduced, e.g. with a certain percentage (e.g. 90% Ala, 3% Ile, 7% Val), and w_(i)'s are assigned weight factors as defined by the user of the program (e.g. depending on the importance of having a specific amino acid residue inserted into the position in question). N is 21 plus the number of amino acid groups as defined by the user of the program. For purposes of this function 0⁰ is defined as being 1.

A Monte-Carlo algorithm (one example being the one described by Valleau, J. P. & Whittington, S. G. (1977) A guide to Mont Carlo for statistical mechanics: 1 Highways. In “Stastistical Mechanics, Part A” Equlibrium Techniqeues ed. B. J. Berne, New York: Plenum) is used for finding the maximum value of this function. In each iteration the following steps are performed:

1. A new random nucleotide composition is chosen for each base, where the absolute difference between the current and the new composition is smaller than or equal to d for each of the four nucleotides G,A,T,C in all three positions of the codon (see below for definition of d).

2. The scores of the new composition and the current composition are compared by the use of the function s as described above. If the new score is higher or equal to the score of the current composition, the new composition is kept and the current composition is changed to the new one. If the new score is smaller, the probability of keeping the new composition is exp(1000(new_score_current_score)).

A cycle normally consists of 1000 iterations as described above in which d is decreasing linearly from 1 to 0. One hundred or more cycles are performed in an optimization process. The nucleotide composition resulting in the highest score is finally presented.

EXAMPLE 1 Construction of Termamyl™ Variants in Accordance with the Invention

Termamyl (B. licheniformis α-amylase SEQ ID NO. 2) is expressed in B. subtilis from a plasmid denoted pDN1528. This plasmid contains the complete gene encoding Termamyl, amyL, the expression of which is directed by its own promoter. Further, the plasmid contains the origin of replication, ori, from plasmid pUB110 and the cat gene from plasmid pC194 conferring resistance towards chloramphenicol. pDN1528 is shown in FIG. 9 of WO 96/23874.

A specific mutagenesis vector containing a major part of the coding region of SEQ ID NO 1 was prepared. The important features of this vector, denoted pJeEN1, include an origin of replication derived from the pUC plasmids, the cat gene conferring resistance towards chloramphenicol, and a frameshift-containing version of the bla gene, the wild type of which normally confers resistance towards ampicillin (amp^(R) phenotype). This mutated version results in an ampS phenotype. The plasmid pJeEN1 is shown in FIG. 10 of WO 96/23874, and the E. coli origin of replication, ori, bla, cat, the 5′-truncated version of the Termamyl amylase gene, and selected restriction sites are indicated on the plasmid.

Mutations are introduced in amyL by the method described by Deng and Nickoloff (1992, Anal. Biochem. 200, pp. 81-88) except that plasmids with the “selection primer” (primer #6616; see below) incorporated are selected based on the amp^(R) phenotype of transformed E. coli cells harboring a plasmid with a repaired bla gene, instead of employing the selection by restriction enzyme digestion outlined by Deng and Nickoloff. Chemicals and enzymes used for the mutagenesis were obtained from the ChameleonÔ mutagenesis kit from Stratagene (catalogue number 200509).

After verification of the DNA sequence in variant plasmids, the truncated gene, containing the desired alteration, is subcloned into pDN1528 as a PstI-EcoRI fragment and transformed into the protease- and amylase-depleted Bacillus subtilis strain SHA273 (described in W092/11357 and W095/10603) in order to express the variant enzyme.

The Termamyl variant V54W was constructed by the use of the following mutagenesis primer (written 5′ to 3′, left to right):

PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG (SEQ ID No. 10)

The Termamyl variant A52W+V54W was constructed by the use of the following mutagenesis primer (written 5′ to 3′, left to right):

PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT CG (SEQ ID No. 11) Primer #6616 (written 5′ to 3′, left to right; P denotes a 5′ phosphate):

P CTG TGA CTG GTG AGT ACT CAA CCA AGT C (SEQ ID No. 12)

The Termamyl variant V54E was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG (SEQ ID No. 13)

The Termamyl variant V54M was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG (SEQ ID No. 14)

The Termamyl variant V54I was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGG TCG TAG GCA CCG TAG CCA ATA TCC GCT TG (SEQ ID No. 15)

The Termamyl variants Y290E and Y290K were constructed by the use of the following mutagenesis primer (written 51-3′, left to right):

PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA C (SEQ ID No. 16) Y represent an equal mixture of C and T. The presence of a codon encoding either Glutamate or Lysine in position 290 was verified by DNA sequencing.

The Termamyl variant N190F was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C (SEQ ID No. 17)

The Termamyl variant N188P+N190F was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC (SEQ ID No. 18)

The Termamyl variant H140K+H142D was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG (SEQ ID No. 19)

The Termamyl variant H156Y was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG (SEQ ID No. 20)

The Termamyl variant A181T was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC (SEQ ID No. 21)

The Termamyl variant A209V was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC (SEQ ID No. 22)

The Termamyl variant Q264S was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC (SEQ ID No. 23)

The Termamyl variant S187D was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC (SEQ ID No. 24)

The Termamyl variant DELTA(K370-G371-D372) (i.e. deleted of amino acid residues nos. 370, 371 and 372) was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC (SEQ ID No. 25)

The Termamyl variant DELTA(D372-S373-Q374) was constructed by the use of the following mutagenesis primer (written 5′-3′, left to right):

PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C (SEQ ID No. 26)

The Termamyl variants A181T and A209V were combined to A181T+A209V by digesting the A181T containing pDN1528-like plasmid (i.e. pDN1528 containing within amyL the mutation resulting in the A181T alteration) and the A209V-containing pDN1528-like plasmid (i.e. pDN1528 containing within amyL the mutation resulting in the A209V alteration) with restriction enzyme ClaI which cuts the pDN1528-like plasmids twice resulting in a fragment of 1116 bp and the vector-part (i.e. contains the plasmid origin of replication) of 3850 bp. The fragment containing the A209V mutation and the vector part containing the A181T mutation were purified by QIAquick gel extraction kit (purchased from QIAGEN) after separation on an agarose gel. The fragment and the vector were ligated and transformed into the protease and amylase depleted Bacillus subtilis strain referred to above. Plasmid from amy+ (clearing zones on starch containing agar-plates) and chloramphenicol resistant transformants were analysed for the presence of both mutations on the plasmid.

In a similar way as described above, H156Y and A209V were combined utilizing restriction endonucleases Acc65I and EcoRI, giving H156Y+A209V.

H156Y +A209V and A181T+A209V were combined into H156Y+A181T+A209V by the use of restriction endonucleases Acc65I and HindIII.

The 35 N-terminal residues of the mature part of Termamyl variant H156Y+A181T+A209V were substituted by the 33 N-terminal residues of the B. amyloliquefaciens α-amylase (SEQ ID NO 4) (which in the present context is termed BAN) by a SOE-PCR approach (Higuchi et al. 1988, Nucleic Acids Research 16:7351) as follows:

Primer 19364 (sequence 5′-3′): CCT CAT TCT GCA GCA GCA GCC GTA AAT GGC ACG CTG (SEQ ID No. 27)

Primer 19362: CCA GAC GGC AGT AAT ACC GAT ATC CGA TAA ATG TTC CG (SEQ ID No. 28)

Primer 19363: CGG ATA TCG GTA TTA CTG CCG TCT GGA TTC (SEQ ID No. 29)

Primer 1C: CTC GTC CCA ATC GGT TCC GTC (SEQ ID No. 30)

A standard PCR, polymerase chain reaction, was carried out using the Pwo thermostable polymerase from Boehringer Mannheim according to the manufacturer's instructions and the temperature cyclus: 5 minutes at 94° C., 25 cycles of (94° C. for 30 seconds, 500C for 45 seconds, 72° C. for 1 minute), 72° C. for 10 minutes.

An approximately 130 bp fragment was amplified in a first PCR denoted PCR1 with primers 19364 and 19362 on a DNA fragment containing the gene encoding the B. amyloliquefaciens α-amylase.

An approximately 400 bp fragment was amplified in another PCR denoted PCR2 with primers 19363 and 1C on template pDN1528.

PCR1 and PCR2 were purified from an agarose gel and used as templates in PCR3 with primers 19364 and 1C, which resulted in a fragment of approximately 520 bp. This fragment thus contains one part of DNA encoding the N-terminus from BAN fused to a part of DNA encoding Termamyl from the 35th amino acid.

The 520 bp fragment was subcloned into a pDN1528-like plasmid (containing the gene encoding Termamyl variant H156Y+A181T+A209V) by digestion with restriction endonucleases PstI and SacII, ligation and transformation of the B. subtilis strain as previously described. The DNA sequence between restriction sites PstI and SacII was verified by DNA sequencing in extracted plasmids from amy+and chloramphenicol resistant transformants.

The final construct containing the correct N-terminus from BAN and H156Y+A181T+A209V was denoted BAN(1-35)+H156Y+A181T+A209V.

N190F was combined with BAN(1-35)+H156Y+A181T+A209V giving BAN(1-35)+H156Y+A181T+N190F+A209V by carrying out mutagenesis as described above exept that the sequence of amyL in pJeEN1 was substituted by the DNA sequence encoding Termamyl variant BAN(1-35)+H156Y+A181T+A209V

Q264S was combined with BAN(1-35)+H156Y+A181T+A209V giving BAN(1-35)+H156Y+A181T+A209V+Q264S by carrying out mutagenesis as described above exept that the sequence of amyL in pJeEN was substituted by the the DNA sequence encoding Termamyl variant BAN(1-35)+H156Y+A181T+A209V

BAN(1-35)+H156Y+A181T+A209V+Q264S and BAN(1-35)+H156Y+A181T+N19OF+A209V were combined into BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S utilizing restriction endonucleases BsaHI (BsaHI site was introduced close to the A209V mutation) and PstI.

EXAMPLE 2 Construction, by Localized Random, Doped Mutagenesis, of Termamyl-like α-Amylase Variants Having an Improved Stability at Low pH and a Reduced Dependency on Calcium Ions for Stability Compared to the Parent Enzyme

α-amylases are of great importance for the industrial starch liquefaction process. The variant of the thermostable B. licheniformis α-amylase consisting of amino acids 1-33 of the B. amyloliquefaciens amylase (SEQ ID NO 4) fused to amino acids 36-483 of the B. licheniformis amylase (SEQ ID NO 2) and further comprising the following mutations:

Y156, T181, F190, V209 and S264 (the construction of this variant is described in Example 1)

has a very satisfactory stability at low pH and low calcium concentrations. In an attempt to further improve the stability at low pH and low calcium concentration of said α-amylase variant random mutagenesis in preselected regions wase performed.

The regions were:

Region: Residue: I: Phe153-Thr163 II: Gln178-Asn192 III: His205-Arg214 IV: Ala232-Asp237 and VIII: Gly131-Lys136 IX: Asp164-Tyr175 X: Tyr262-Thr278 Region changed Total % Mean % Number of residues I: 35 88  8 out of 11 II: 20 86 11 out of 15 III: 27 88 10 out of 10 IV: 34 91 11 out of 12 VIII: 39 86  6 out of 6 IX: 46 93 12 out of 12 X: 27 90 12 out of 13 VIII + IX: 18 VIII + IX + II: 4 II + III + IV: 2 IV + I: 12

The numbers under Total % give the total number of wild-type (wt) amino acids desired in a given region after doping. The number is obtained by multiplication of the number of mutated positions (e.g. 8 with respect to region I) by their respective wt. With respect to region I the desired total % is 80*80*90*90*90*90*95*90/100=35%.

The Mean % is the mean doping level for the total number of positions of the region in question (e.g. 11 positions with respect to region I). For region I the mean % is calculated as follows: 80+80+90+90+90+90+95+90=705 divided by 11=88%

The DOPE software (see Materials and Methods) was used to determine spiked codons for each suggested change in the seven regions minimizing the amount of stop codons. The exact distribution of nucleotides was calculated in the three positions of the codon to give the suggested population of amino acid changes. The doped regions were doped specifically in the indicated positions to have a high chance of getting the desired residues, but still allow other possibilities.

For instance, the original H156 in the wt sequence was mutated into an Y, meaning a new codon, and then doped 10% for other residues. That is the DNA sequence has the code for a Y instead for a H. In position 156 the Tyr has been programmed to be 90% desired and other residues has been freely allowed. For some positions it was not possible to create the suggested population of amino acid residues because the genetic code restricted the structurally and functionally desired residues. The resulting seven doped oligonucleotides are shown in tables 1-7: with the wt nucleotide and amino acid sequences and the distribution of nucleotides for each doped position. All library primers were synthesized as sense strands.

TABLE 1 Library DASI (Phe153-Thr163) 153 154 155 156 157 158 159 160 161 162 163 Phe Lys Trp Tyr Trp Tyr His Phe Asp Gly Thr Primer: 5′CGC GGC AGC ACA TAC AGC GAT T1T 2A3 TGG 45T TGG 67T 8AT TTT GAC GGA A9C GAT TGG GAC GAG TCC CGA AAG3′ (SEQ ID NO.31) Distribution of nucleotides for each doped position. 1: 80% T, 20% A. 2: 96% A, 2% G, 2% C. 3: 98% A, 2% T. 4: 93% T, 4% G, 3% A. 5: 97% A, 3% G. 6: 98% T, 2% A. 7: 97% A, 3% C. 8: 90% C, 10% T. 9: 95% C, 5% A.

TABLE 2 Library DASII (Gln178-Asn192) 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 Gln Gly Lys Thr Trp Asp Trp Glu Val Ser Asn Glu Phe Gly Asn Primer: 5′CTG AAC CGC ATC TAT AAG TTT 1A2 34T AAG 567 TGG GAT 89G GA10 GTT A11T 1213T GAA T1415 161718 AAC TAT GAT TAT TTG ATG TAT3′ (SEQ ID NO.32) Distribution of nucleotides for each doped position.  1: 93% C, 7% A.  2: 84% G, 16% T.  3: 95% G, 5% A.  4: 95% G, 5% C.  5: 94% A, 6% G.  6: 95% C, 5% A.  7: 62% T, 38% G.  8: 87% T, 13% A.  9: 91% G, 9% C. 10: 92% G, 8% T. 11: 90% G, 5% A, 5% C. 12: 88% A, 12% C. 13: 88% A, 12% C. 14: 93% T, 5% A, 2% C. 15: 97% T, 3% G. 16: 86% G, 14% A. 17: 89% G, 11% C. 18: 60% G, 40% T.

TABLE 3 Library DASIII (His205-Arg214) 205 206 207 208 209 210 211 212 213 214 His Pro Asp Val Val Ala Glu Ile Lys Arg Primer: 5′TAT GCC GAC ATC GAT TAT GAC 12T 3CT 456 7TT 8910 1112T 13A14 15T16 A17A 1819A TGG GGC ACT TGG TAT GCC AAT 3′ (SEQ ID NO.33) Distribution of nucleotides for each doped position.  1: 89% C, 11% T.  2: 89% A, 11% G.  3: 95% C, 2.5% T, 2.5% A.  4: 96% G, 1% A, 3% T.  5: 96% A, 4% C.  6: 98% T, 2% A.  7: 95% G, 2.5% A, 2.5% C.  8: 93% G, 7% A.  9: 96% T, 4% A. 10: 84% A, 16% G. 11: 81% G, 7% A, 7% T, 5% C. 12: 98% C, 2% A. 13: 96% G, 4% C. 14: 94% G, 6% T. 15: 82% A, 18% T. 16: 50% A, 50% T. 17: 90% A, 10% G. 18: 70% A, 30% C. 19: 86% G, 14% A

TABLE 4 Library DASIV (Ala232-Asp243) 232 233 234 235 236 237 238 239 240 241 242 243 Ala Val Lys His Ile Lys Phe Ser Phe Leu Arg Asp Primer: 5′TTG GAC GGT TTC CGT CTT GAT 12T G3T AAA 456 7TT A8G T9T 1011T T12T 13T14 1516G GA17 TGG GTT AAT CAT GTC AGG GAA (SEQ ID NO.34) Distribution of nucleotides for each doped position.  1: 93% G, 3.5% A, 3.5% T.  2: 94% C, 4% T.  3: 94% T, 6% C.  4: 93% C, 2% T, 2% A, 3% G.  5: 98% A, 2% T.  6: 98% T, 2% A.  7: 95% A, 5% C.  8: 94% A, 6% G.  9: 90% T, 10% A. 10: 89% T, 11% A. 11: 89% C, 11% A. 12: 95% T, 5% A 13: 64% C, 33% T, 3% A. 14: 93% A, 7% T. 15: 90% A, 10% C. 16: 90% G, 5% A, 5% C 17: 90% T, 10% A

TABLE 5 Library DASVIII (Gly131-Lys136) 131 132 133 134 135 136 Gly Glu His Leu Ile lys Primer: 5′ GCT GAC CGC AAC CGC GTA ATT TCA 123 GA4 56T 78A 9TA A10G GCC TGG ACA CAT TTT CAT TTT 3′ (SEQ ID NO.35) Distribution of nucleotides for each doped position.  1: 91% G, 9% A.  2: 87% G, 13% C.  3: 90% T, 10% G.  4: 90% G, 10% T.  5: 85% C, 8% T, 7% A.  6: 89% A, 9% T, 2% C.  7: 88% T, 12% A  8: 88% T, 11% C, 1% G  9: 92% A, 8% T 10: 93% A, 7% G

TABLE 6 Library DASIX (Asp164-Tyr175) 164 165 166 167 168 169 170 171 172 173 174 175 Asp Trp Asp Glu Ser Arg lys Leu Asn Arg Ile Tyr Primer: 5′TGG TAC CAT TTT GAC GGA ACC GAT TGG 1A2 GAG 3CG CGA A4G 56A A7T A8G 9 1011 T12T AAG TTT CAA GGA AAG GCT TGG 3′ (SEQ ID NO.36) Distribution of nucleotides for each doped position.  1: 94% G, 6% A.  2: 96% T, 4% G.  3: 92% T, 4% A, 4% G.  4: 95% A, 5% G.  5: 93% C, 7% A.  6: 92% T, 8% A.  7: 90% A, 5% G, 5% C.  8: 90% G, 10% A.  9: 92% A, 6% G, 2% T. 10: 92% T, 8% A. 11: 50% T, 50% C. 12: 96% A, 4% T.

TABLE 7 Library DASX (Tyr262-Asn278) 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 Tyr Trp Ser Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Primer: 5′GAA ATG TTT ACG GTA GCT GAA T1T TGG 234 56T 7A8 91011 1213T 1415T 16T17 GA18 A19T T20T 21T22 A23C A24G ACA 25AT TTT AAT CAT TCA GTG TTT GAC3′ (SEQ ID NO.37) Distribution of nucleotides for each doped position.  1: 95% A, 5% T.  2: 97% A, 3% G.  3: 95% G, 2.5% A, 2.5% C.  4: 94% T, 6.2% G.  5: 97% A, 3% T.  6: 94% A, 3% G, 3% C.  7: 95% G, 5% A.  8: 95% T, 5% A.  9: 52% T, 45% C, 3% A. 10: 96% T, 4% C. 11: 60% A, 40% G. 12: 90% G, 10% A. 13: 94% G, 6% C. 14: 81% G, 8% A, 8% T, 3% C. 15: 98% C, 2% T. 16: 90% C, 10% A. 17: 50% G, 50% T. 18: 90% A, 10% T. 19: 90% A, 5% G, 5% C. 20: 95% A, 5% T. 21: 91% T, 9% A. 22: 92% A, 8% G. 23: 94% A, 3% G, 3% C. 24: 93% G, 7% A. 25: 90% A, 10% G.

Random Mutagenesis

The spiked oligonucleotides apparent from Tables 1-7 (which by a common term is designated FDAS in FIG. 3) and reverse primers RDAS for each region and specific B. licheniformis primers covering the SacII and the SalI sites are used to generate PCR-library-fragments by the overlap extension method (Horton et al., Gene, 77 (1989), pp. 61-68) with an overlap of 21 bp. FIG. 3 shows the PCR strategy. The PCR fragments are cloned in the E. coli/Bacillus shuttle vector pSNK101 (see Materials and Methods) enabling mutagenesis in E. coli and immediate expression in Bacillus subtilis preventing lethal accumulation of amylases in E. coli. After establishing the cloned PCR fragments in E. coli, a modified pUC19 fragment is digested out of the plasmid and the promoter and the mutated Termamyl gene is physically connected and expression can take place in Bacillus.

Screening

The seven libraries may be screened in the low pH and the low calcium filter assays described below.

Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 μg/ml chloramphenicol at 37° C. for at least 21 hrs. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with citrate buffer, pH 4.5 and incubated at 80° C. for 15 min. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hrs. at 50° C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.

Low Calcium Filter Assay

The assay is performed in the same way as the low pH filter assay with the following modifications: The filter with bound proteins is incubated at 95° C., pH 6.0 for 1 h. with different EDTA concentrations (0.001 mM-100 mM).

The following variants were obtained by the above method (BAN designates B. amyloliquefaciens α-amylase):

BAN/Termamyl hybrid *+H156Y+A181T+N190F+A209V+Q264S+E211Q

BAN/Termamyl hybrid *+H156Y+A181T+N190F+A209V+Q264S+H205C+D207Y+A210S

The mutations indicated in bold were introduced by the random mutagenesis method. The stability data for these variants appear from Table 11 in Example 3.

In an analogous manner to that described above, random mutagenesis of the above identified seven regions are performed on the parent B. licheniformis α-amylase (SEQ ID NO 2). The doping scheme is determined analogously to that used above.

EXAMPLE 3 Measurement of the Calcium- and pH-dependent Stability

Normally, the industrial liquefaction process runs using pH 6.0-6.2 as liquefaction pH and an addition of 40 ppm free calcium in order to improve the stability at 95° C.-105° C . Some of the herein proposed substitutions have been made in order to improve the stability at

1. lower pH than pH 6.2 and/or

2. at free calcium levels lower than 40 ppm free calcium.

Three different methods have been used to measure the improvements in stability obtained by the different substitutions in Termamyl:

1. One assay which measures the stability at slightly reduced pH, pH 5.5, in the presence of 4 ppm free calcium. (thereby, the improvement of stability at low pH is measured). 10 μg of the variant were incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 5.5, containing 40 ppm calcium and 5% w/w common corn starch (free of calcium). Incubation was made in a water bath at 95° C. for 30 minutes.

2. Another assay which measure the stability in the absence of free calcium and where the pH is maintained at pH 6.2. This assay measures the decrease in calcium sensitivity:

10 μg of the variant were incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 6.2, containing 5% w/w common corn starch (free of calcium). Incubation was made in a water bath at 95° C. for 30 minutes.

3. A third assay wherein the conditions of assays nos. 1 and 2 have been combined. This assay measures the stability in the absence of calcium and at low pH (pH 5.5).

4. A fourth assay similar to no.3. where the pH has been further reduced to pH 5,0

Stability Determination

All the stability trials 1,2, 3 and 4 have been made using the same set up. The method was:

The enzyme was incubated under the relevant conditions (1-4). Samples were taken at 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity was measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) was used as reference (100%). The decline in percent was calculated as a function of the incubation time. The table shows the residual activity after 30 minutes of incubation.

Specific Activity Determination

The specific activity was determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The activity was determined using the α-amylase assay described in the Materials and Methods section herein.

Results:

TABLE 8 Stability method no.1. / Low pH stability improvement residual activity Specific activity, after 30 min. of 37° C., pH 7.3. Percent Variant incubation relative to Termamyl Termamyl wt.  5% 100% H156Y 15% 100% A209V 18% 100% Q264S 30% 130% H156Y + A181T + A209V 39% 100% H133Y + H156Y + A181T + A209V + 58%  85% H293Y + A294V + G310D + H450Y BAN/Termamyl hybrid * + 48% 115% H156Y + A181T + A209V

TABLE 9 Stabi1ity method no.2. / decreased calcium sensitivity residual activity Specific activity, after 30 min. of 37° C., pH 7.3. Percent Variant incubation relative to Termamyl Termamyl wt.  52% — H156Y 70% — A209V 75% — A181T 57% 115% N190F 34% 140% N190F + N188P 57% 190%

TABLE 10 Stability method no.3. / Low pH stability improvement + decreased calcium sensitivity residual activity Specific activity, after 30 min. of 37° C., pH 7.3. Variant incubation Units/mg enzyme Termamyl wt.  3% — BAN/Termamyl hybrid * + 20% — H156Y + A181T + A209V Q264S  5% — H140K + H142D  5% 115% V128E   50% ** 115% BAN/Termamyl hybrid * + 39% 170% H156Y + A181T + N190F + A209V BAN/Termamyl hybrid * + 29% 175% H156Y + A181T + A209V + Q264S BAN/Termamyl hybrid * + 57% 210% H156Y + A181T + N190F + A209V + Q264S * BAN (B. amyloliquefaciens α-amylase (SEQ ID NO 4)/Termamyl (B. licheniformis α-amylase (SEQ ID NO 2) PCR hybrid. First 33 N-terminal aminoacids are BAN and the rest 36-483 are Termamyl (the construction of the variant is described in Example 1). ** Measured after 5 min. of incubation. Compared to Termamyl wt. Which under same conditions shows 36% residual activity.

TABLE 11 Stability method no.4. / Low pH stability improvement (pH 5,0) + decreased calcium sensitivity residual activity Specific activity, after 30 min. of 37° C., pH 7.3. Variant incubation Units/mg enzyme BAN/Termamyl hybrid * +  9% 210% H156Y + A181T + N190F + A209V + Q264S BAN/Termamyl hybrid * + 28% 160% H156Y + A181T + N190F + A209V + Q264S + E211Q BAN/Termamyl hybrid * + 33% 130% H156Y + A181T + N190F + A209V + Q264S + H205C + D207Y + A210S * as indicated in relation to Table 10

The variants in the above Table 11 were constructed by means of the localized random mutagenesis described in Example 2.

EXAMPLE 5 α-Amylase Stability at Low pH and High Temperature

This example summarises the stability results of variants characterised by a fluorimetric assay at 70° C. under two different conditions, (1) pH 4.5 and 1 mM CaCl₂ and (2) pH 6.2 and 10 μM CaCl₂.

Description of Method

All fluorescence experiments were performed on a Perkin-Elmer LS-50 luminescence spectrometer using a 4-cuvette holder. The temperature was controlled by a circulating water-bath and measured directly in the cuvette using a Noronix Digital Thermometer (model NTD 100). During measurements, thorough mixing of reagents in the cuvette was ensured using magnetic stirrers operating at high stirring rate. The cuvettes were capped with teflon-lids to minimize evaporation.

Intrinsic protein-fluorescence (due to Trp side-chains) was monitored by excitation at 280 nm and emission at 350 nm. Slit-widths were 5 nm.

During kinetic measurements, 4 reactions were monitored in parallel. Data was collected in the Wavelength Programme dialogue, allowing automatic data-collection over a prolonged period (e.g. over an hour).

Unfolding was carried out at 70° C. Unfolding conditions were

(1) 50 mM NaOAc pH 4.5 and 1 mM CaCl₂

(2) 50 mM NaOAc pH 6.2 and 10 AM CaCl₂. Protein concentration was 5 μg/ml and glycerol was at 0.5% w/v (from protein stock solution).

Note: There was some variation from day to day in the absolute value of the unfolding half times due to slight temperature variations (occasioned by e.g. different amounts of water in the water bath). However, Termamyl was always included as one of the four enzymes analyzed in each experiment, in effect making it an internal standard. Unfolding rates relative to this internal standard were satisfactorily reproducible (performed in triplicate). Data analysis was carried out using GraphPad Prism software. At pH 4.5, unfolding data could be fitted very satisfactorily to a single-exponential decay with drift:

F(t)=A*exp(−ln(2)*t/t _(½))+drift*t+offset  (1)

where F is the measured fluorescence, A is the amplitude of the unfolding, t is time and t_(½) is the half-time of unfolding.

At pH 6.2, unfolding was more complex (involving an initial lag phase), and data could not be fitted to eq. 1. Instead, the time taken for the fluorescence signal to decay to 50% of the initial signal was used as an apparent t_(½).

From these half-times, the change in free energy of unfolding relative to that of Termamyl could be calculated as follows: $\begin{matrix} {{DDG} = {R*T*{\ln \left( {t_{1/2}^{mutant}/t_{1/2}^{Termanyl}} \right)}}} & (2) \end{matrix}$

where R is the universal gas constant and T is the temperature (the value of R*T is 0.5919, giving a DDG value in kcal/mol).

By converting data to DDG values, the destabilizing/stabilizing effects of different mutations can be compared directly and examined for additivity (DDG₁₊₂=DDG₁+DDG₂) and synergy (DDG₁₊₂>DDG₁+DDG₂) where DDG₁₊₂ is the energy-effect of introducing mutations 1 and 2.

Results

Unfolding of amylases at low pH and high temperature may be followed by the decay in Trp-fluorescence. At pH 4.5 and 1 mM CaCl2, all amylases unfold fairly rapidly.

The unfolding data at pH 4.5 fit better to a double-exponential equation than to a single-exponential equation. However, since the second phase is very slow, it is approximated by a linear drift (equation 1). Unfolding at pH 6.2 and 10 μM CaCl₂ at 70° C. is much less rapid than at pH 4.5 despite the low [Ca²⁺]. Unfolding is far from complete within an hour and it is not possible to fit the data to a single-exponential equation. Instead, the time taken for the fluorescence signal to decay to 50% of the initial signal is used as an apparent t_(½).

Results of the fluorescence assay are presented in Table 12.

TABLE 12 Summary of data for unfolding of Termamyl variants pH 4.5 and pH 6.2 at 70° C. At pH 4.5, t_(1/2) ^(Termamyl) = 200 s; at pH 6.2, t_(1/2) ^(Termamyl) = 2800 s. DDG = −RT*1n (t_(1/2) ^(Termamyl)/t_(1/2) ^(mutant)) pH 4.5, 1 mM CaCl₂ pH 6.2, 10 μM CaCl₂ DDG DDG Mutation (kcal/mol) t_(1/2)/t_(1/2) ^(Termamyl) (kcal/mol) t_(1/2)/t_(1/2) ^(Termamyl) Wildtype 0 1.0 0 1.0 A209V −0.36 1.85 −0.72 3.39 H133Y + A209V −0.77 3.67 −0.61 2.78 H156Y 0.06 0.90 −0.10 1.18 A181T −0.06 1.10 −0.28 1.60 A181T + A209V −0.44 2.09 <−1 (73%)^(a) >5 S187D 0.37 0.54 0.19 0.72 H450Y −0.49 2.29 0.15 0.78 L270D −0.35 1.8 −0.10 1.2 A181T + H156Y −0.17 1.34 −0.62 2.84 H133I −0.33 1.75 −0.42 2.02 H133Y + H156Y + A181T + −0.96 5.10 <−1 (58%)^(a) >5 A209V + H293Y + A294V G310D + H450Y V128E −0.10 1.2 −0.25 1.5 H156Y + A181T + A209V −0.32 1.71 −0.49 2.30 H156Y + A181T + A209V + −0.42 2.05 −0.63 2.92 H450Y H156Y + A181T + A209V + −0.81 3.9 <−1 (65%)^(a) >5 H450Y + H133I H156Y + A181T + A209V + −0.70 3.3 <−1 (77%)^(a) >5 H133I Q264S −0.26 1.6 −0.14 1.3 H156Y + A181T + A209V + −0.43 2.1 −0.82 4.0 Delta (1,2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I Q264S + N265Y −0.33 1.8 −0.07 1.1 Q264S + N265Y + N190F −1.07 6.1 −0.67 3.1 Q264S + N265Y + N190F + −1.66 16.5 <−1 (82%)^(a) >5 H133I + A209V H156Y + A181T + A209V + −0.30 1.7 <−1 (66%)^(a) >5 Delta (1,2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I + N190F H156Y + A181T + A209V + −0.43 2.1 −0.86 4.3 Delta (1,2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I + Q264S H156Y + A181T + A209V + −0.36 1.8 <−1 (76%)^(a) >5 Delta (1,2) + L3V + M15T + R23K + S31A + A32E + Y33H + A35S + E36D + H37I Q264S + N190F H156Y + A181T + A209V + −1.3 8.6 <−1 (66%)^(a) >5 N190F + Q264S ^(a)The percentage indicates the level to which the initial fluorescence level had declined in the course of 3 hours at 70 + 20 + 0 C. The slow decline is indicative of high stability.

EXAMPLE 5 α-Amylase Variants with Increased Specific Activity

This example summarises the results of variants characterised by having increased specific activity compared to Termamyl wt. The presence of these substitutions either in combination with each other or as single substitutions added to stabilising substitutions increases the specific activity of the resulting variant. The specific activity was determined using the α-amylase (Phadebas) assay described in the Materials and Methods where the activity/mg enzyme was determined. The activity was determined using the following description where the pH was 7.3, temperature 37° C. and testing time 15 min. and buffer as defined.

SPEC. ACTIVITY (PHADEBAS ASSAY), INDEX RELATIVE MUTATION TO TERMAMYL WT. S187D 260% V541 160% BAN/Termamyl hybrid: 140% (Δ(1,2)+L3V+M15T+R23K+S31A+ A32E+Y33H+A35S+E36D+H371) Δ(D372+S373+Q374) 125% Δ (K370+G371+D372) 125% BAN/Termamyl hybrid: 360% (Δ (1, 2) +L3V+M15T+R23K+S31A+ A32E+Y33H+A35S+E36D+H371)+ Δ(D372+S373+Q374) +V541+S187D Q264S 130% Y290E 155% Y290K 140% N19OF 150%

EXAMPLE 6 Testing of Specificity Variants (Saccharification)

It has been reported previously (U.S. Pat. No. 5,234,823) that, when saccharifying with glucoamylase and pullulanase, the presence of residual α-amylase activity arising from the liquefaction process, can lead to lower yields of glucose, if the α-amylase is not inactivated before the saccharification stage. This inactivation can be typically carried out by adjusting the pH to below 4.3 at 95° C., before lowering the temperature to 60° C. for saccharification.

The reason for this negative effect on glucose yield is not fully understood, but it is assumed that the liquefying α-amylase (for example Termamyl 120 L from B.licheniformis) generates “limit dextrins” (which are poor substrates for pullulanase), by hydrolysing 1,4-alpha-glucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build up of the trisaccharide panose, which is only slowly hydrolysed by glucoamylase.

The development of a thermostable α-amylase, which does not suffer from this disadvantage would be a significant improvement, as no separate inactivation step would be required.

A number of B.licheniformis α-amylase variants, with altered specificity, were evaluated by saccharifying a DE 10 Maltodextrin substrate with A.niger glucoamylase and B.acidopullulyticus pullulanase under conditions where the variant amylase was active.

The saccharification reactions were monitored by taking samples at 24 hour intervals and analysing them by HPLC. The standard reaction conditions were:

Substrate concentration 28.2% W/W Temperature 60° C.C Initial pH (at 60° C.) 4.7 Enzyme dosage Glucoamylase 0.18 AG/g DS pullulanase O.06 PUN/g DS α-amylase 60 NU/g DS

The following enzymes were used:

Glucoamylase: AMG (Nava Nordisk) 153 AG/g Pullulanase: Promozyme (Novo Nordisk) 295 PUN/g α-amylase: Termamyl (Novo Nordisk) 135 KNU/g V54Y 313 KNU/g A52W 5,116 NU/ml D53E 3,280 NU/ml D53W 599 NU/ml A52W+V54Y 134 NU/ml

The mutations listed in the α-amylase list above are used to indicate variants of the B. licheniformis α-amylase (SEQ ID NO 2) (Termamyl) which has been modified by the indicated mutation(s).

Substrates for saccharification were prepared by dissolving 230 g DE 10 spray-dried maltodextrin, prepared from common corn starch, in 460 ml boiling deionized water and adjusting the dry substance to approximately 30% w/w. The pH was adjusted to 4.7 (measured at 60° C.) and aliquots of substrate corresponding to 15 g dry weight, were transferred to 50 ml blue cap glass flasks.

The flasks were then placed in a shaking water bath equilibrated at 60° C., and the enzymes added. The pH was readjusted to 4.7 where necessary. 2 ml samples were taken periodically, the pH adjusted to about 3.0, and then heated in a boiling water bath for 15 minutes to inactivate the enzymes. After cooling, the samples were treated with approximately 0.1 g mixed bed ion exchange resin (BIO-Rad 501 X8 (D)) for 30 minutes on a rotary mixer to remove salts and soluble N. After filtration, the carbohydrate composition was determined by HPLC. After 72 hours, the following results were obtained:

Added α-amylase % DP₁ % DP₂ % DP₃ % DP₄₊ None (control) 96.59 2.2 0.3 1.0 V54Y 96.5 2.2 0.4 0.9 A52W+V54Y 96.4 2.2 0.5 0.9 Termamyl 96.3 2.1 0.8 0.8

Compared with the control (no active α-amylase present during liquefaction), the presence of active α-amylase variants V54Y and A52W+V54Y did not lead to elevated panose levels (DP₃).

If these α-amylase variants are used for starch liquefaction, it will not be necessary to inactivate the enzyme before the commencement of saccharification.

EXAMPLE 7 Evaluation of B.licheniformis Variants Under Simulated Liquefaction Conditions

The standard process for industrial starch liquefaction comprises two stages, normally referred to as primary and secondary liquefaction. In the first stage, a 30-40% w/w starch slurry at pH 5.5-6.0, to which has been added a thermostable alpha-amylase from B. licheniformis or B. stearothermophillus, is heated to 105-110° C. in a jet cooker where live steam is injected into the starch stream. After a holding time of 5-10 minutes under pressure at this temperature, the liquefied starch is flash cooled to about 95° C. and held at that temperature for 60-120 minutes.

In order to evaluate small quantities of enzyme on a laboratory scale the following test method was used:

10 g aliquots of a suspension of common corn starch (Cerestar GL 3406) in deionized water (approx. 30% w/w) are weighed out into 100 ml conical flasks (Schott GL 125) which are fitted with tight fitting screw caps. The pH, calcium level and enzyme dosage in the suspension can be varied.

4 flasks are used for each different set of experimental conditions. The flasks are placed in a shaking oil-bath (Heto VS 01) maintained at 105° C. After a period of 7 minutes, cold oil is poured into the bath to lower the temperature to 95° C. For each experimental series, flasks are removed after 20, 40, 60 and 90 minutes and immediately cooled under running water. One drop of 1N HCL is added to each flask to inactivate the enzyme.The reaction is monitored by measuring the DE (reducing sugar content expressed as glucose) using the Neocuproine method.

The details of this method can be found in “Determination of reducing sugar with improved precision. Dygert, Li, Florida and Thomas, Anal Biochem, 13, 368 (1965).

The following DEs were recorded after 90 minutes

Hybrid + Hybrid + H156Y + Hybrid + H156Y + H156Y + A181T + H156Y + Hybrid + A181T + A181T + A209V + A181T + H156Y + Q264S + H156Y + A209V + A209V + N190F + A209V + A181T + Q264S + N265Y + A181T + I.D. no. Termamyl N190F + Q264S N190 F Q264S Q264S A209V Q264S N265Y N190F A209V pH 6.0 5 ppm calcium added 10.9 mg enzyme protein/g starch 045-96 16.0 16.9 13.2 038-96 6.5 13.9 035-96 15.2 12.9 9.9 033-96 6.7 7.2 12.1 031-96 4.5 7.0 8.8 12.5 029-96 4.0 8.7 5.2 7.7 039-96 14.9 16.3 14.4 pH 6.0 40 ppm calcium added 10.9 mg enzyme protein/g starch 045-96 12.6 007-96 12.1 pH 5.5 5 ppm calcium added 10.9 mg enzyme protein/g starch 001-97 14.8 15.2 12.6 Hybrid = BAN/Termamyl PCR hybrid as described in Example 3

REFERENCES CITED

Klein, C., et al., Biochemistry 1992, 31, 8740-8746,

Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,

Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238,

Larson, S. B., J. Mol. Biol. (1994) 235, 1560-1584,

Lawson, C. L., J. Mol. Biol. (1994) 236, 590-600,

Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,

Brady, R. L., et al., Acta Crystallogr. sect. B, 47, 527-535,

Swift, H. J., et al., Acta Crystallogr. sect. B, 47, 535-544

A. Kadziola, Ph.D. Thesis: “An alpha-amylase from Barley and its Complex with a Substrate Analogue Inhibitor Studied by X-ray Crystallography”, Department of Chemistry University of Copenhagen 1993

MacGregor, E. A., Food Hydrocolloids, 1987, Vol.1, No. 5-6, p.

B. Diderichsen and L. Christiansen, Cloning of a maltogenic α-amylase from Bacillus stearothermophilus, FEMS Microbiol. letters: 56: pp. 53-60 (1988)

Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications,

Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989

S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869

Matthes et al., The EMBO J. 3, 1984, pp. 801-805.

R. K. Saiki et al., Science 239, 1988, pp. 487-491.

Morinaga et al., (1984, Biotechnology 2:646-639)

Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151

Hunkapiller et al., 1984, Nature 310:105-111

R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl. Acids Res. 16:7351-7367.

Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.

Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329. S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.

Boel et al., 1990, Biochemistry 29, pp. 6244-6249.

37 1 1920 DNA Bacillus licheniformis 1 cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gcaaatctta atgggacgct gatgcagtat tttgaatggt acatgcccaa tgacggccaa 480 cattggaggc gtttgcaaaa cgactcggca tatttggctg aacacggtat tactgccgtc 540 tggattcccc cggcatataa gggaacgagc caagcggatg tgggctacgg tgcttacgac 600 ctttatgatt taggggagtt tcatcaaaaa gggacggttc ggacaaagta cggcacaaaa 660 ggagagctgc aatctgcgat caaaagtctt cattcccgcg acattaacgt ttacggggat 720 gtggtcatca accacaaagg cggcgctgat gcgaccgaag atgtaaccgc ggttgaagtc 780 gatcccgctg accgcaaccg cgtaatttca ggagaacacc taattaaagc ctggacacat 840 tttcattttc cggggcgcgg cagcacatac agcgatttta aatggcattg gtaccatttt 900 gacggaaccg attgggacga gtcccgaaag ctgaaccgca tctataagtt tcaaggaaag 960 gcttgggatt gggaagtttc caatgaaaac ggcaactatg attatttgat gtatgccgac 1020 atcgattatg accatcctga tgtcgcagca gaaattaaga gatggggcac ttggtatgcc 1080 aatgaactgc aattggacgg tttccgtctt gatgctgtca aacacattaa attttctttt 1140 ttgcgggatt gggttaatca tgtcagggaa aaaacgggga aggaaatgtt tacggtagct 1200 gaatattggc agaatgactt gggcgcgctg gaaaactatt tgaacaaaac aaattttaat 1260 cattcagtgt ttgacgtgcc gcttcattat cagttccatg ctgcatcgac acagggaggc 1320 ggctatgata tgaggaaatt gctgaacggt acggtcgttt ccaagcatcc gttgaaatcg 1380 gttacatttg tcgataacca tgatacacag ccggggcaat cgcttgagtc gactgtccaa 1440 acatggttta agccgcttgc ttacgctttt attctcacaa gggaatctgg ataccctcag 1500 gttttctacg gggatatgta cgggacgaaa ggagactccc agcgcgaaat tcctgccttg 1560 aaacacaaaa ttgaaccgat cttaaaagcg agaaaacagt atgcgtacgg agcacagcat 1620 gattatttcg accaccatga cattgtcggc tggacaaggg aaggcgacag ctcggttgca 1680 aattcaggtt tggcggcatt aataacagac ggacccggtg gggcaaagcg aatgtatgtc 1740 ggccggcaaa acgccggtga gacatggcat gacattaccg gaaaccgttc ggagccggtt 1800 gtcatcaatt cggaaggctg gggagagttt cacgtaaacg gcgggtcggt ttcaatttat 1860 gttcaaagat agaagagcag agaggacgga tttcctgaag gaaatccgtt tttttatttt 1920 2 483 PRT Bacillus licheniformis 2 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Arg 3 2084 DNA Bacillus amyloliquefaciens 3 gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc 120 atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180 ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240 agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc 300 acgctgttat ttgtcagttt gccgattaca aaaacatcag ccgtaaatgg cacgctgatg 360 cagtattttg aatggtatac gccgaacgac ggccagcatt ggaaacgatt gcagaatgat 420 gcggaacatt tatcggatat cggaatcact gccgtctgga ttcctcccgc atacaaagga 480 ttgagccaat ccgataacgg atacggacct tatgatttgt atgatttagg agaattccag 540 caaaaaggga cggtcagaac gaaatacggc acaaaatcag agcttcaaga tgcgatcggc 600 tcactgcatt cccggaacgt ccaagtatac ggagatgtgg ttttgaatca taaggctggt 660 gctgatgcaa cagaagatgt aactgccgtc gaagtcaatc cggccaatag aaatcaggaa 720 acttcggagg aatatcaaat caaagcgtgg acggattttc gttttccggg ccgtggaaac 780 acgtacagtg attttaaatg gcattggtat catttcgacg gagcggactg ggatgaatcc 840 cggaagatca gccgcatctt taagtttcgt ggggaaggaa aagcgtggga ttgggaagta 900 tcaagtgaaa acggcaacta tgactattta atgtatgctg atgttgacta cgaccaccct 960 gatgtcgtgg cagagacaaa aaaatggggt atctggtatg cgaatgaact gtcattagac 1020 ggcttccgta ttgatgccgc caaacatatt aaattttcat ttctgcgtga ttgggttcag 1080 gcggtcagac aggcgacggg aaaagaaatg tttacggttg cggagtattg gcagaataat 1140 gccgggaaac tcgaaaacta cttgaataaa acaagcttta atcaatccgt gtttgatgtt 1200 ccgcttcatt tcaatttaca ggcggcttcc tcacaaggag gcggatatga tatgaggcgt 1260 ttgctggacg gtaccgttgt gtccaggcat ccggaaaagg cggttacatt tgttgaaaat 1320 catgacacac agccgggaca gtcattggaa tcgacagtcc aaacttggtt taaaccgctt 1380 gcatacgcct ttattttgac aagagaatcc ggttatcctc aggtgttcta tggggatatg 1440 tacgggacaa aagggacatc gccaaaggaa attccctcac tgaaagataa tatagagccg 1500 attttaaaag cgcgtaagga gtacgcatac gggccccagc acgattatat tgaccacccg 1560 gatgtgatcg gatggacgag ggaaggtgac agctccgccg ccaaatcagg tttggccgct 1620 ttaatcacgg acggacccgg cggatcaaag cggatgtatg ccggcctgaa aaatgccggc 1680 gagacatggt atgacataac gggcaaccgt tcagatactg taaaaatcgg atctgacggc 1740 tggggagagt ttcatgtaaa cgatgggtcc gtctccattt atgttcagaa ataaggtaat 1800 aaaaaaacac ctccaagctg agtgcgggta tcagcttgga ggtgcgttta ttttttcagc 1860 cgtatgacaa ggtcggcatc aggtgtgaca aatacggtat gctggctgtc ataggtgaca 1920 aatccgggtt ttgcgccgtt tggctttttc acatgtctga tttttgtata atcaacaggc 1980 acggagccgg aatctttcgc cttggaaaaa taagcggcga tcgtagctgc ttccaatatg 2040 gattgttcat cgggatcgct gcttttaatc acaacgtggg atcc 2084 4 480 PRT Bacillus amyloliquefaciens 4 Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 5 1814 DNA Bacillus stearothermohilus 5 aaattcgata ttgaaaacga ttacaaataa aaattataat agacgtaaac gttcgagggt 60 ttgctccctt tttactcttt ttatgcaatc gtttccctta attttttgga agccaaaccg 120 tcgaatgtaa catttgatta agggggaagg gcattgtgct aacgtttcac cgcatcattc 180 gaaaaggatg gatgttcctg ctcgcgtttt tgctcactgt ctcgctgttc tgcccaacag 240 gacagcccgc caaggctgcc gcaccgttta acggcaccat gatgcagtat tttgaatggt 300 acttgccgga tgatggcacg ttatggacca aagtggccaa tgaagccaac aacttatcca 360 gccttggcat caccgctctt tggctgccgc ccgcttacaa aggaacaagc cgcagcgacg 420 tagggtacgg agtatacgac ttgtatgacc tcggcgaatt caatcaaaaa gggaccgtcc 480 gcacaaaata cggaacaaaa gctcaatatc ttcaagccat tcaagccgcc cacgccgctg 540 gaatgcaagt gtacgccgat gtcgtgttcg accataaagg cggcgctgac ggcacggaat 600 gggtggacgc cgtcgaagtc aatccgtccg accgcaacca agaaatctcg ggcacctatc 660 aaatccaagc atggacgaaa tttgattttc ccgggcgggg caacacctac tccagcttta 720 agtggcgctg gtaccatttt gacggcgttg attgggacga aagccgaaaa ttgagccgca 780 tttacaaatt ccgcggcatc ggcaaagcgt gggattggga agtagacacg gaaaacggaa 840 actatgacta cttaatgtat gccgaccttg atatggatca tcccgaagtc gtgaccgagc 900 tgaaaaactg ggggaaatgg tatgtcaaca caacgaacat tgatgggttc cggcttgatg 960 ccgtcaagca tattaagttc agtttttttc ctgattggtt gtcgtatgtg cgttctcaga 1020 ctggcaagcc gctatttacc gtcggggaat attggagcta tgacatcaac aagttgcaca 1080 attacattac gaaaacagac ggaacgatgt ctttgtttga tgccccgtta cacaacaaat 1140 tttataccgc ttccaaatca gggggcgcat ttgatatgcg cacgttaatg accaatactc 1200 tcatgaaaga tcaaccgaca ttggccgtca ccttcgttga taatcatgac accgaacccg 1260 gccaagcgct gcagtcatgg gtcgacccat ggttcaaacc gttggcttac gcctttattc 1320 taactcggca ggaaggatac ccgtgcgtct tttatggtga ctattatggc attccacaat 1380 ataacattcc ttcgctgaaa agcaaaatcg atccgctcct catcgcgcgc agggattatg 1440 cttacggaac gcaacatgat tatcttgatc actccgacat catcgggtgg acaagggaag 1500 ggggcactga aaaaccagga tccggactgg ccgcactgat caccgatggg ccgggaggaa 1560 gcaaatggat gtacgttggc aaacaacacg ctggaaaagt gttctatgac cttaccggca 1620 accggagtga caccgtcacc atcaacagtg atggatgggg ggaattcaaa gtcaatggcg 1680 gttcggtttc ggtttgggtt cctagaaaaa cgaccgtttc taccatcgct cggccgatca 1740 caacccgacc gtggactggt gaattcgtcc gttggaccga accacggttg gtggcatggc 1800 cttgatgcct gcga 1814 6 514 PRT Bacillus stearothermophilus 6 Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp 7 478 PRT Bacillus licheniformis 7 Ala Thr Pro Ala Asp Trp Arg Ser Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp Arg Phe Ala Arg Thr Asp Gly Ser Thr Thr Ala Thr Cys Asn Thr 20 25 30 Ala Asp Gln Lys Tyr Cys Gly Gly Thr Trp Gln Gly Ile Ile Asp Lys 35 40 45 Leu Asp Tyr Ile Gln Gly Met Gly Phe Thr Ala Ile Trp Ile Thr Pro 50 55 60 Val Thr Ala Gln Leu Pro Gln Thr Thr Ala Tyr Gly Asp Ala Tyr His 65 70 75 80 Gly Tyr Trp Gln Gln Asp Ile Tyr Ser Leu Asn Glu Asn Tyr Gly Thr 85 90 95 Ala Asp Asp Leu Lys Ala Leu Ser Ser Ala Leu His Glu Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val Val Ala Asn His Met Gly Tyr Asp Gly Ala 115 120 125 Gly Ser Ser Val Asp Tyr Ser Val Phe Lys Pro Phe Ser Ser Gln Asp 130 135 140 Tyr Phe His Pro Phe Cys Phe Ile Gln Asn Tyr Glu Asp Gln Thr Gln 145 150 155 160 Val Glu Asp Cys Trp Leu Gly Asp Asn Thr Val Ser Leu Pro Asp Leu 165 170 175 Asp Thr Thr Lys Asp Val Val Lys Asn Glu Trp Tyr Asp Trp Val Gly 180 185 190 Ser Leu Val Ser Asn Tyr Ser Ile Asp Gly Leu Arg Ile Asp Thr Val 195 200 205 Lys His Val Gln Lys Asp Phe Trp Pro Gly Tyr Asn Lys Ala Ala Gly 210 215 220 Val Tyr Cys Ile Gly Glu Val Leu Asp Gly Asp Pro Ala Tyr Thr Cys 225 230 235 240 Pro Tyr Gln Asn Val Met Asp Gly Val Leu Asn Tyr Pro Ile Tyr Tyr 245 250 255 Pro Leu Leu Asn Ala Phe Lys Ser Thr Ser Gly Ser Met Asp Asp Leu 260 265 270 Tyr Asn Met Ile Asn Thr Val Lys Ser Asp Cys Pro Asp Ser Thr Leu 275 280 285 Leu Gly Thr Phe Val Glu Asn His Asp Asn Pro Arg Phe Ala Ser Tyr 290 295 300 Thr Asn Asp Ile Ala Leu Ala Lys Asn Val Ala Ala Phe Ile Ile Leu 305 310 315 320 Asn Asp Gly Ile Pro Ile Ile Tyr Ala Gly Gln Glu Gln His Tyr Ala 325 330 335 Gly Gly Asn Asp Pro Ala Asn Arg Glu Ala Thr Trp Leu Ser Gly Tyr 340 345 350 Pro Thr Asp Ser Glu Leu Tyr Lys Leu Ile Ala Ser Ala Asn Ala Ile 355 360 365 Arg Asn Tyr Ala Ile Ser Lys Asp Thr Gly Phe Val Thr Tyr Lys Asn 370 375 380 Trp Pro Ile Tyr Lys Asp Asp Ile Thr Ile Ala Met Arg Lys Gly Thr 385 390 395 400 Asp Gly Ser Gln Ile Val Thr Ile Leu Ser Asn Lys Gly Ala Ser Gly 405 410 415 Asp Ser Tyr Thr Leu Ser Leu Ser Gly Ala Gly Tyr Thr Ala Gly Gln 420 425 430 Gln Leu Thr Glu Val Ile Gly Cys Thr Thr Val Thr Val Gly Ser Asp 435 440 445 Gly Asn Val Pro Val Pro Met Ala Gly Gly Leu Pro Arg Val Leu Tyr 450 455 460 Pro Thr Glu Lys Leu Ala Gly Ser Lys Ile Cys Ser Ser Ser 465 470 475 8 20 DNA Bacillus amyloliquefaciens 8 gacctgcagt caggcaacta 20 9 20 DNA Bacillus amyloliquefaciens 9 tagagtcgac ctgcaggcat 20 10 31 DNA Bacillus licheniformis 10 ggtcgtaggc accgtagccc caatccgctt g 31 11 36 DNA Bacillus licheniformis 11 ggtcgtaggc accgtagccc caatcccatt ggctcg 36 12 28 DNA Bacillus licheniformis 12 ctgtgactgg tgagtactca accaagtc 28 13 31 DNA Bacillus licheniformis 13 ggtcgtaggc accgtagccc tcatccgctt g 31 14 31 DNA Bacillus licheniformis 14 ggtcgtaggc accgtagccc atatccgctt g 31 15 31 DNA Bacillus licheniformis 15 ggtcgtaggc accgtagcca atatccgctt g 31 16 36 DNA Bacillus licheniformis 16 gcagcatgga actgctyatg aagaggcacg tcaaac 36 17 30 DNA Bacillus licheniformis 17 catagttgcc gaattcattg gaaacttccc 30 18 34 DNA Bacillus licheniformis 18 catagttgcc gaattcaggg gaaacttccc aatc 34 19 41 DNA Bacillus licheniformis 19 ccgcgccccg ggaaatcaaa ttttgtccag gctttaatta g 41 20 32 DNA Bacillus licheniformis 20 caaaatggta ccaataccac ttaaaatcgc tg 32 21 29 DNA Bacillus licheniformis 21 cttcccaatc ccaagtcttc ccttgaaac 29 22 36 DNA Bacillus licheniformis 22 cttaatttct gctacgacgt caggatggtc ataatc 36 23 38 DNA Bacillus licheniformis 23 cgcccaagtc attcgaccag tactcagcta ccgtaaac 38 24 29 DNA Bacillus licheniformis 24 gccgttttca ttgtcgactt cccaatccc 29 25 35 DNA Bacillus licheniformis 25 ggaatttcgc gctgactagt cccgtacata tcccc 35 26 36 DNA Bacillus licheniformis 26 ggcaggaatt tcgcgacctt tcgtcccgta catatc 36 27 36 DNA Bacillus amyloliquefaciens 27 cctcattctg cagcagcagc cgtaaatggc acgctg 36 28 38 DNA Bacillus amyloliquefaciens 28 ccagacggca gtaataccga tatccgataa atgttccg 38 29 30 DNA Bacillus amyloliquefaciens 29 cggatatcgg tattactgcc gtctggattc 30 30 21 DNA Bacillus amyloliquefaciens 30 ctcgtcccaa tcggttccgt c 21 31 75 DNA Bacillus licheniformis 31 cgcggcagca catacagcga ttwtvawtgg drttggwmty attttgacgg aamcgattgg 60 gacgagtccc gaaag 75 32 84 DNA Bacillus licheniformis 32 ctgaaccgca tctataagtt tmakrstaag rmktgggatw sggakgttav tmmtgaathk 60 rskaactatg attatttgat gtat 84 33 72 DNA Bacillus licheniformis misc_feature (0)...(0) N at 37 is 81% G, 7% A, 7% T, 5% C 33 tatgccgaca tcgattatga cyrthctdmw vttrwrnmts akwtwaramr atggggcact 60 tggtatgcca at 72 34 78 DNA Bacillus licheniformis misc_feature (0)...(0) N at 31 is 93% C, 2% T, 2% A, 3% G 34 ttggacggtt tccgtcttga tdytgytaaa nwwmttargt wtwmttwtht wmvggawtgg 60 gttaatcatg tcagggaa 78 35 63 DNA Bacillus licheniformis 35 gctgaccgca accgcgtaat ttcarskgak hhtwbawtaa rggcctggac acattttcat 60 ttt 63 36 78 DNA Bacillus licheniformis 36 tggtaccatt ttgacggaac cgattggrak gagdcgcgaa rgmwaavtar gdwytwtaag 60 tttcaaggaa aggcttgg 78 37 93 DNA Bacillus licheniformis misc_feature (0)...(0) N at 43 is 81% G, 8% T, 3% C 37 gaaatgttta cggtagctga atwttggrvk wvtrawhyrr stnytmtkga wavttwtwtr 60 avcargacar attttaatca ttcagtgttt gac 93 

What is claimed is:
 1. A DNA sequence encoding a variant of a parent Termamyl-like alpha-amylase, wherein said variant has alpha-amylase activity and comprises at least two substitutions at positions corresponding to positions in the amino acid sequence of SEQ ID NO:2 selected from the group consisting of: H68, H247, H382, H450, N172, N188; N190, A209, A210, Q264, and N265.
 2. A recombinant expression vector comprising a DNA construct as defined in claim
 1. 3. A cell transformed with a vector as defined in claim
 2. 4. A cell as defined in claim 3, wherein said cell is a microorganism.
 5. A cell as defined in claim 3, wherein said cell is a bacterium or a fungus.
 6. A cell as defined in claim 3, wherein said cell is a gram positive bacterium selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus and Bacillus thuringiensis. 